Oncoprotein protein kinase

ABSTRACT

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

This application is a continuation application of U.S. application Ser.No. 09/461,649 filed Dec. 14, 1999, now issued as U.S. Pat. No.6,342,595; which is a continuation application of U.S. application Ser.No. 09/150,201 filed Sep. 8, 1998, now issued as U.S. Pat. No.6,001,584; which is a divisional application of U.S. application Ser.No. 08/799,913 filed Feb. 13, 1997, now issued as U.S. Pat. No.5,804,399; which is a continuation application of U.S. application Ser.No. 08/444,393 filed May 19, 1995, now issued as U.S. Pat. No.5,605,808; which is a divisional application of U.S. application Ser.No. 08/276,860 filed Jul. 18, 1994, now issued as U.S. Pat. No.5,593,884; which is a continuation-in-part application of U.S.application Ser. No. 08/220,602 filed Mar. 25, 1994, now issued as U.S.Pat. No. 6,514,745; which is a continuation-in-part application of U.S.application Ser. No. 08/094,533 filed Jul. 19, 1993, now issued as U.S.Pat. No. 5,534,426.

This invention was made with support by Howard Hughes Medical Instituteand Government support under Grant No. DE-86ER60429, awarded by theDepartment of Energy and Grant No. CA-50528 and CA-58396, awarded by theNational Institute of Health. The Government has certain rights in thisinvention. Also supported by the Howard Hughes Medical Institute.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of protein kinases,oncogenes and oncoproteins and, specifically, to a protein kinase whichbinds, phosphorylates and potentiates the c-Jun N-terminal activationdomain.

2. Description of Related Art

A number of viral and cellular genes have been identified as potentialcancer genes, collectively referred to as oncogenes. The cellularhomologs of viral oncogenes, the proto-oncogenes or c-oncogenes, act inthe control of cell growth and differentiation or mediate intracellularsignaling systems. The products of oncogenes are classified according totheir cellular location, for example, secreted, surface, cytoplasmic,and nuclear oncoproteins.

Proto-oncogenes which express proteins which are targeted to the cellnucleus make up a small fraction of oncogenes. These nuclearproto-oncoproteins typically act directly as transactivators andregulators of RNA and DNA synthesis. Nuclear oncogene products have theability to induce alterations in gene regulation leading to abnormalcell growth and ultimately neoplasia. Examples of nuclear oncogenesinclude the myc, ski, myb, fos and jun genes.

The c-Jun protein, encoded by the c-jun proto-oncogene, is an importantcomponent of the dimeric, sequence specific, transcriptional activator,AP-1. Like other transcriptional activators, c-Jun contains twofunctional domains, including a DNA binding domain and a transactivationdomain. The DNA binding domain is located at the C-terminus and is aBZip structure which consists of conserved basic (B) and leucine zipper(Zip) domains that are required for DNA binding and dimerization,respectively. The N-terminus contains the transactivation domain.Although c-Jun expression is rapidly induced by many extracellularsignals, its activity is also regulated post-translationally by proteinphosphorylation. Phosphorylation of sites clustered next to c-Jun's DNAbinding domain inhibits DNA binding (Boyle, et al., Cell, 64:573, 1991;Lin, et al., Cell, 70:777, 1992). Phosphorylation of two other sites,Ser 63 and Ser 73, located within the transactivation domain,potentiates c-Jun's ability to activate transcription (Binetruy, et al.,Nature 351:122, 1991; Smeal, et al., Nature 354:494, 1991).Phosphorylation rates of these sites are low in non-stimulated cells andare rapidly increased in response to growth factors such as plateletderived growth factor (PDGF) or v-Sis, or expression of oncogenicallyactivated Src, Ras and Raf proteins. In myeloid and lymphoid cells,phosphorylation of these sites is stimulated by the phorbol ester, TPA,but not in fibroblasts and epithelial cells. These differences may bedue to different modes of Ha-ras regulation in lymphoid cells versusfibroblasts.

Many proteins cooperate with each other in the activation oftranscription from specific promoters. Through this cooperation, a genecan be transcribed and a protein product generated. Members of the Fosproto-oncogene family, along with members of the Jun gene family, formstable complexes which bind to DNA at an AP-1 site. The AP-1 site islocated in the promoter region of a large number of genes. Binding ofthe Fos/Jun complex activates transcription of a gene associated with anAP-1 site. In cells that have lost their growth regulatory mechanisms,it is believed that this Fos/Jun complex may “sit” on the AP-1 site,causing overexpression of a particular gene. Since many proliferativedisorders result from the overexpression of an otherwise normal gene,such as a proto-oncogene, it would be desirable to identify compositionswhich interfere with the excessive activation of these genes.

For many years, various drugs have been tested for their ability toalter the expression of genes or the translation of their messages intoprotein products. One problem with existing drug therapy is that ittends to act indiscriminately and affects healthy cells as well asneoplastic cells. This is a major problem with many forms ofchemotherapy where there are severe side effects primarily due to theaction of toxic drugs on healthy cells.

In view of the foregoing, there is a need to identify specific targetsin the abnormal cell which are associated with the overexpression ofgenes whose expression products are implicated in cell proliferativedisorders, in order to decrease potential negative effects on healthycells. The present invention provides such a target.

SUMMARY OF THE INVENTION

The present invention provides a novel protein kinase (JNK) whichphosphorylates the c-Jun N-terminal activation domain. JNK1 ischaracterized by having a molecular weight of 46 kD (as determined byreducing SDS-polyacrylamide gel electrophoresis (PAGE)) and havingserine and threonine kinase activity. Specifically, JNK1 phosphorylatesserine residues 63 and 73 of c-Jun.

Since the product of the jun proto-oncogene is a transactivator proteinwhich binds at AP-1 sites, regulation of c-Jun activation may beimportant in affecting normal gene expression and growth control in acell. The discovery of JNK provides a means for identifying compositionswhich affect JNK activity, thereby affecting c-Jun activation andsubsequent activation of genes associated with AP-1 sites.

The identification of JNK now allows the detection of the level ofspecific kinase activity associated with activation of c-Jun and AP-1.In addition, the invention provides a method of treating a cellproliferative disorder associated with JNK by administering to a subjectwith the disorder, a therapeutically effective amount of a reagent whichmodulates JNK activity.

The invention also provides a synthetic peptide comprising the JNKbinding region on c-Jun which corresponds to amino acids 33-79. Thepeptide is useful as a competitive inhibitor of the naturally occurringc-Jun in situations where it is desirable to decrease the amount ofc-Jun activation by JNK.

The invention also describes JNK2, a novel protein kinase with activitysimilar to JNK1 and having a molecular weight of 55 kD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an SDS-PAGE of nuclear and cytosolic extracts from FR3T3(−) and Ha-ras-transformed FR3T3 (+) cells after incubation with ³²P-ATPand GST-cJun (wt), GSTcJun(Ala63/73) or GST.

FIG. 2 shows an SDS-PAGE of A) HeLaS3 cells either untreated orirradiated with UV light and B) Jurkat cells either untreated orincubated with TPA. Cell extracts were incubated with ³²P-ATP andGST-cJun (wt), GSTcjun-(Ala63/73) or GST.

FIG. 3 shows phosphopeptide mapping of GST-cJun and c-Jun phosphorylatedby JNK. 3(A) shows maps of GSTcJun and (B) shows maps of c-Jun.

FIG. 4A shows an SDS-PAGE of phosphorylated proteins after elution ofJNK from GSTc-Jun after washes of NaCl, Urea, Guanidine-HCl(GuHCl) orSDS.

FIG. 4B shows an SDS-PAGE of phosphorylated c-Jun after GSTcJun(wt) wascovalently linked to GSH-beads and incubated with whole cell extract ofTPA-stimulated Jurkat cells.

FIG. 5 shows an in-gel kinase assay. GSTcJun-GSH agarose beads wereincubated with cell extracts from A) TPA-stimulated Jurkat cells on SDSgels that were polymerized in the absence (−) or presence (+) of GSTcJun(wt); B) extracts of unstimulated or UV stimulated HeLa cells andunstimulated or TPA-stimulated Jurkat cells; and C) extracts from cellsof logarithmically growing K562 and Ha-ras-transformed FR3T3,TPA-stimulated Jurkat and U937 cells and UV-irradiated HeLa, F9 and QT6cells.

FIG. 6A is a protein gel of various GST c-Jun fusion proteins;

FIG. 6B shows an SDS-PAGE of whole cell extracts of UV-irradiated HelaS3 cells after passage over GSH-beads containing the GST fusion proteinsas shown in FIG. 6A;

FIG. 6C shows an SDS-PAGE of phosphorylated GSTcJun fusion proteinseluted with 1MNaCl from GSH-agarose beads.

FIG. 7A shows patterns of GST, GSTcJun and GSTvJun as expressed in Ecoli;

FIG. 7B shows the phosphorylated proteins of 7A from extracts ofTPA-activated Jurkat cells incubated with GSH-beads;

FIG. 7C shows cJun protein after phosphorylation with protein bound toGSTcJun and GSTvJun beads.

FIG. 8 shows CAT activity in cells containing various portions of thec-Jun activation domain (cJ=AA1-223; 33=AA33-223; 43=AA43-223;56=AA56-223; A63,73=AA1-246(Ala63/73)) and a CAT reporter in the absenceor presence of A) Ha-ras or B) UV treatment.

FIG. 9 shows SDS-PAGE analyses of ³²P and ³⁵S labelled F9 cellstransfected with v-Jun and c-Jun in the absence or presence of A) Ha-rasor B) UV exposure.

FIGS. 10A-10E shows the nucleotide and deduced amino acid sequence ofc-Jun. The arrows represent amino acid residues 33-79.

FIG. 11A shows a Northern blot of total cytoplasmic RNA from Jurkatcells. Cells were incubated with 50 ng/ml TPA (T), 1 μg/ml A23187 (A) or100 ng/ml cyclosporin A (CsA) for 40 minutes, either alone or incombination, as indicated. Levels of c-jun, jun-B, jun-D, c-fos andα-tubulin expression were determined by hybridization to random primedcDNA probes.

FIG. 11B shows Jurkat cells after incubatation with soluble anti-CD3(OKT3), 2 μg/ml soluble anti-CD28 (9.3) or a combination of 50 ng/ml TPAand 1 μg/ml A23817 (T/A) as indicated for 40 minutes. Total cytoplasmicRNA was isolated and 10 μg samples were analyzed using c-jun, jun-D andc-fos probes. IL-2 induction by the same treatments was measured after 6hours of stimulation by blot hybridization using IL-2 and α-tubulinspecific probes.

FIG. 11C shows Jurkat cells transfected with 10 μg of either −73Col-LUCor −60Col-LUC reporter plasmids. 24 hours after transfection, the cellswere aliquoted into 24 well plates and incubated for 9 hours with 50ng/ml TPA, 1 μg/ml A23187 or 100 ng/ml CsA, either alone or incombination, as indicated. The cells were harvested and luciferaseactivity was determined. The results shown are averages of threeexperiments done in triplicates.

FIG. 12A shows Jurkat cells (10⁶ cells per lane) transfected with 0.5 μgof a SRα-cJun expression vector and 24 hours later were labeled for 3hours with ³²P-orthophosphate (1 mCi/ml). After 15 minutes, treatmentwith 50 ng/ml TPA (T), 1 μg/ml A23187 (A) and 100 ng/ml CsA, eitheralone or in combination, as indicated, the cells were lysed in RIPAbuffer and c-Jun was isolated by immunoprecipitation and analyzed bySDS-PAGE. The c-Jun bands are indicated.

FIG. 12B shows 2×10⁷ Jurkat cells labeled for 3 hours with either³⁵S-methionine (900 μCi/ml) or ³²P-orthophosphate (1 mCi/ml). After 15minutes incubation with 50 ng/ml TPA+1 μg/ml A23178 (T/A) in the absenceor presence of and 100 ng/ml CsA or no addition, as indicated, the cellswere lysed in RIPA buffer and c-Jun isolated by immunoprecipitation andanalyzed by SDS-PAGE. The c-Jun band is indicated.

FIG. 12C shows all of the c-Jun specific protein bands shown in FIG. 12Aisolated from equal numbers of cells excised from the gel and subjectedto tryptic phosphopeptide mapping. Shown is a typical result (thisexperiment was repeated at least three times). N-nonstimulated cells;T-cells treated with 50 ng/ml TPA; T/A: cells treated with 50 ng/ml TPAand 1 μg/ml A23187; T/A+CsA: cells treated with T/A and 100 ng/ml CsA.a,b,c,x and y correspond to the various tryptic phosphopeptides ofc-Jun, previously described by Boyle, et al., (Cell, 64:573-584, 1991)and Smeal, et al., (Nature, 354:494-496, 1991). T1 and T2 correspond tothe minor phosphorylation sites; Thr91, 93 and 95 (Hibi, et al., Genes &Dev., 7:000, 1993).

FIG. 13A shows whole cell extracts (WCE) of Jurkat cells incubated withTPA (T, 50 ng/ml), A23187 (A, 1 μg/ml) or CsA (100 ng/ml) for 15minutes, alone or in combination, and separated by SDS-PAGE (100 μgprotein/lane) on gels that were cast in the absence or presence ofGST-cJun (1-223). The gels were subjected to renaturation protocol andincubated in kinase buffer containing γ-³²P-ATP. The protein bandscorresponding to the 55 kD and 46 kD forms of JNK are indicated.

FIG. 13B shows WCE (50 μg) of Jurkat cells treated as described abovewere incubated with 5 μl of GSH agarose beads coated with 10 μg GST-cJun(1-223) for 12 hours at 4° C. After extensive washing, the beads wereincubated in kinase buffer containing γ-³²P-ATP for 20 minutes at 30°C., after which the proteins were dissociated by incubation in SDSsample buffer and separated by SDS-PAGE. The 49 kD band corresponds toGST-cJun (1-223).

FIG. 13C shows WCE (200 μg) of Jurkat cells treated as described in FIG.13A and incubated with GST-cJun(1-223)-GSH agarose beads. The boundfraction was eluted in SDS sample buffer and separated by SDS-PAGE on agel containing GST-cJun(1-223). The gel was renatured and incubated inkinase buffer containing γ-³²P-ATP to label the JNK polypeptides.

FIG. 14 shows a phosphorylation assay of cultures of FR3T3, CV-1, PC12and mouse thymocytes were incubated for 15 minutes in the presence ofTPA (50 ng/ml, T), A23817 (1 μg/ml, A) and/or CsA (100 ng/ml), asindicated. WCE prepared from 2-4×10⁵ cells for the established celllines and 1.5×10⁶ cells for primary thymocytes were incubated withGSTcJun(1-223)-GSH agarose beads. After washing, JNK activity wasdetermined by solid-state phosphorylation assay.

FIG. 15 shows WCE (5 μg) of Jurkat (panel A) or mouse thymocytes (panelC) incubated with 1 μg of kinase-defective ERK1 in kinase buffercontaining γ-³²P-ATP for 20 minutes. The phosphorylated proteins wereseparated by SDS-PAGE and the band corresponding to the mutant ERK1 isindicated. WCE (20 μg) of Jurkat (panel A) or mouse thymocytes (panel C)that were treated as described above were immunoprecipitated withanti-ERK antibodies. The immune complexes were washed and incubated inkinase buffer containing γ-³²P-ATP and 2 μg MBP for 15 minutes at 30° C.The phosphorylated proteins were separated by SDS-PAGE. The bandcorresponding to phosphorylated MBP is indicated in panels B and D.

FIG. 16A shows Jurkat cells (1×10⁷) incubated for 15 minutes with eithernormal mouse serum, 1 μg/ml anti-CD3 and/or 2 μg/ml anti-CD28, in theabsence or presence of 100 ng/ml CsA, as indicated. WCE were preparedand 100 μg samples were analyzed for JNK activation using an in-gelkinase assay.

FIG. 16B shows WCE (50 μg) of Jurkat cells treated as described for FIG.16A incubated with GSTcJun(1-223)-GSH agarose beads and assayed for JNKactivity using the solid-state kinase assay. The same WCE (20 μg) wereimmunoprecipitated with anti-ERK2 antibodies and assayed for MBP-kinaseactivity.

FIG. 16C shows WCE (50 μg) of Jurkat cells treated as described in FIG.16A with various stimuli alone or their combinations were incubated withGSTcJun(1-223)-GSH agarose beads and assayed for JNK activity usingsolid-state kinase assay. The same samples (20 μg) were also assayed forMBP-kinase activity as described in FIG. 16B.

FIG. 17A shows Jurkat cells (2×10⁶ cells per point) labeled with 0.4 mCiof ³²P-orthophosphate for 3 hours and incubated with nonspecificantibody (1 μg/ml mouse IgG; control), 1 μg/ml anti-CD3, 2 μg/mlanti-CD28, 10 ng/ml TPA or 500 ng/ml A23187 (A), as indicated. After 2minutes, the cells were harvested, lysed and Ha-Ras was isolated byimmunoprecipitation. The guanine nucleotide bound to Ha-Ras wasextracted, separated by thin layer chromatography and quantitiated. Thevalues shown represent the averages of two separate experiments done induplicates.

FIG. 17B shows Jurkat cells labeled with ³²P-orthophosphate andstimulated with either TPA or anti-CD3. At the indicated time points,the cells were harvested and the GTP content of Ha-Ras was determined.

FIGS. 18A and 18D show the nucleotide and deduced amino acid sequence ofJNK1.

FIG. 18B shows a comparison of the deduced sequence of JNK1 with otherMAP kinases.

FIG. 18C shows a comparison of the deduced structure of JNK1 with theGenBank data-base.

FIG. 19A shows a Northern blot analysis of JNK1 in fetal brain.

FIG. 19B shows a Northern blot analysis of JNK1 in adult tissues.

FIG. 19C shows a Southern blot analysis of human genomic DNA hybridizedwith a JNK1 probe.

FIG. 20A shows JNK1 kinase activity as measured in an SDS-PAGE using anin-gel kinase assay with GST-c-Jun (1-79) substrate.

FIG. 20B shows a time course of JNK1 protein kinase activation by EGFand TPA.

FIGS. 20C and 20D show the time course and dose response of JNK1activation by UV radiation.

FIGS. 20E and 20F show the dose response of JNK1 UV-induced activation.

FIGS. 21A and 21B shows a time course and dose response of UV activationof the endogenous JNK1 protein kinase expressed by COS cells.

FIGS. 22A and 22B show an immunocomplex kinase assay with substrateGST-c-Jun (1-79) to show the effect of Ha-Ras and UV on JNK1 activity.

FIG. 23 shows immunoprecipitation studies of JNK1 expressed in COS cellsand activated by UV (panel A), in Hela cells (panel B), or a mixture ofpurified ERK1 and ERK2 (panel C). The Coomasie blue stain of the proteinsubstrates is shown in panel D.

FIGS. 24A, 24B, 24C, 24D, and 24E show phosphopeptide map mapping ofc-Jun phosphorylated by JNK-46.

FIG. 25A shows phosphorlated GST-c-Jun proteins detected by a solidstate protein kinase assay.

FIG. 25B shows Western blot analysis of epitope-tagged JNK1 expressed inCOS cells.

FIG. 26 show analyses of UV-stimulated phosphorylation of JNK1 aftersubstitution of Thr-183 or Tyr-185. Panel A & B show Western blotanalysis using chemiluminescence detection or cells metabolicallylabeled with ³²P-phosphate, respectively. Panel C shows phosphoaminoacid analysis. Panel D shows an SDS-PAGE using an in-gel kinase assaywith the substrate GST-c-Jun (1-79).

FIGS. 27A and 27B show the results of tryptic phosphopeptide mapping ofJNK1 purified from F9 cells transfected with epitope tagged JNK1 cells.

FIG. 28 is the nucleotide and deduced amino acid sequence of JNK2.

FIG. 29 is the deduced amino acid sequence of JNK2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel protein kinase (JNK) which bindsto a well-defined region of the c-Jun proto-oncoprotein andphosphorylates two sites within its activation domain. Thephosphorylation of these sites increases the ability of c-Jun tostimulate transcription and mediate oncogenic transformation.

The activity of c-Jun is regulated by phosphorylation. Various stimuli,including transforming oncogenes and UV light, induce thephosphorylation of serines 63 and 73 in c-Jun's N-terminal activationdomain, thereby potentiating its transactivation function. The inventionrelates to an isolated polypeptide characterized by having a molecularweight of 46 kD as determined by reducing SDS-PAGE, having serine andthreonine kinase activity and capable of phosphorylating the c-JunN-terminal activation domain. This protein is referred to JNK1. Inaddition, a second JNK protein (55 kD) referred to as JNK2, is described

The term “isolated” means any JNK polypeptide of the present invention,or any gene encoding a JNK polypeptide, which is essentially free ofother polypeptides or genes, respectively, or of other contaminants withwhich the JNK polypeptide or gene might normally be found in nature.

The invention includes a functional polypeptide, JNK, and functionalfragments thereof. As used herein, the term “functional polypeptide”refers to a polypeptide which possesses a biological function oractivity which is identified through a defined functional assay andwhich is associated with a particular biologic, morphologic, orphenotypic alteration in the cell. The biological function, for example,can vary from a polypeptide fragment as small as an epitope to which anantibody molecule can bind to a large polypeptide which is capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An enzymatically functionalpolypeptide or fragment of JNK possesses c-Jun N-terminal activationdomain kinase activity. A “functional polynucleotide” denotes apolynucleotide which encodes a functional polypeptide as describedherein.

Minor modifications of the JNK primary amino acid sequence may result inproteins which have substantially equivalent activity as compared to theJNK polypeptide described herein. Such modifications may be deliberate,as by site-directed mutagenesis, or may be spontaneous. All of thepolypeptides produced by these modifications are included herein as longas the kinase activity of JNK is present. Further, deletion of one ormore amino acids can also result in a modification of the structure ofthe resultant molecule without significantly altering its kinaseactivity. This can lead to the development of a smaller active moleculewhich would have broader utility. For example, it is possible to removeamino or carboxy terminal amino acids which may not be required for JNKkinase activity.

The JNK polypeptide of the invention also includes conservativevariations of the polypeptide sequence. The term “conservativevariation” as used herein denotes the replacement of an amino acidresidue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

The invention also provides a synthetic peptide which binds to the c-JunN-terminal kinase, JNK. The amino acid sequence of SEQ ID NO: 1, andconservative variations, comprises the synthetic peptide of theinvention. This sequence represents amino acids 33-79 of c-Junpolypeptide (Angel, et al., Nature, 332(6160):166, 1988) As used herein,the term “synthetic peptide” denotes a peptide which does not comprisean entire naturally occurring protein molecule. The peptide is“synthetic” in that it may be produced by human intervention using suchtechniques as chemical synthesis, recombinant genetic techniques, orfragmentation of whole antigen or the like.

Peptides of the invention can be synthesized by such commonly usedmethods as t-BOC or FMOC protection of alpha-amino groups. Both methodsinvolve stepwise syntheses whereby a single amino acid is added at eachstep starting from the C terminus of the peptide (See, Coligan, et al.,Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well knownsolid phase peptide synthesis methods described Merrifield, J. Am. Chem.Soc., 85:2149, 1962), and Stewart and Young, Solid Phase PeptidesSynthesis, (Freeman, San Francisco, 1969, pp.27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on Sephadex G-15 using 5%acetic acid as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

The invention also provides polynucleotides which encode the JNKpolypeptide of the invention and the synthetic peptide of SEQ ID NO: 1.As used herein, “polynucleotide” refers to a polymer ofdeoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct. DNA encoding thepolypeptide of the invention can be assembled from cDNA fragments orfrom oligonucleotides which provide a synthetic gene which is capable ofbeing expressed in a recombinant transcriptional unit. Polynucleotidesequences of the invention include DNA, RNA and cDNA sequences.Preferably, the nucleotide sequence encoding JNK1 is the sequence of SEQID NO: 11 and JNK2 is the sequence in FIG. 28.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences; 2) antibody screening of expression libraries todetect shared structural features and 3) synthesis by the polymerasechain reaction (PCR).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the poiypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

The development of specific DNA sequences encoding JNK can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA. Of these three methods for developingspecific DNA sequences for use in recombinant procedures, the isolationof genomic DNA isolates is the least common. This is especially truewhen it is desirable to obtain the microbial expression of mammalianpolypeptides due to th e presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay et al., Nucl. Acid Res. 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for JNK polypeptide having at least one epitope, usingantibodies specific for JNK. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of JNK cDNA.

A polynucleotide sequence can be deduced from the genetic code, however,the degeneracy of the code must be taken into account. Polynucleotidesof the invention include sequences which are degenerate as a result ofthe genetic code. The polynucleotides of the invention include sequencesthat are degenerate as a result of the genetic code. There are 20natural amino acids, most of which are specified by more than one codon.Therefore, as long as the amino acid sequence of JNK results in afunctional polypeptide (at least, in the case of the sensepolynucleotide strand), all degenerate nucleotide sequences are includedin the invention.

The polynucleotide sequence for JNK also includes sequencescomplementary to the polynucleotide encoding JNK (antisense sequences).Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). The invention embraces all antisensepolynucleotides capable of inhibiting production of JNK polypeptide. Inthe cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. The antisense nucleic acidsinterfere with the translation of the mRNA since the cell will nottranslate a mRNA that is double-stranded. Antisense oligomers of about15 nucleotides are preferred, since they are easily synthesized and areless likely to cause problems than larger molecules when introduced intothe target JNK-producing cell. The use of antisense methods to inhibitthe translation of genes is well known in the art (Marcus-Sakura,Anal.Biochem., 172:289, 1988).

In addition, ribozyme nucleotide sequences for JNK are included in theinvention. Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med.-Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that that sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The JNK polypeptides of the invention can also be used to produceantibodies which are immunoreactive or bind to epitopes of the JNKpolypeptides. Antibodies of the invention also include antibodies whichbind to the synthetic peptide in SEO ID NO: 1. Antibody which consistsessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known in the art (Kohler, etal., Nature, 256:495, 1975; Current Protocols in Molecular Biology,Ausubel, et al., ed., 1989).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)₂, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to the JNK polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide such as Sequence ID No.1 used to immunize an animal can bederived from translated cDNA or chemical synthesis which can beconjugated to a carrier protein, if desired. Such commonly used carrierswhich are chemically coupled to the peptide include keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanustoxoid. The coupled peptide is then used to immunize the animal (e.g., amouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991, incorporated by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody. Thus, inthe present invention, an anti-idiotype antibody produced from anantibody which binds to the synthetic peptide of the invention can bindto the site on JNK which binds to c-Jun, thereby preventing JNK frombinding to and phosphorylating c-Jun.

Polynucleotide sequences encoding the polypeptide (SEQ ID NO:12 and FIG.29) or the synthetic peptide (SEQ ID NO: 1) of the invention can beexpressed in either prokaryotes or eukaryotes. Hosts can includemicrobial, yeast, insect and mammalian organisms. Methods of expressingDNA sequences having eukaryotic or viral sequences in prokaryotes arewell known in the art. Biologically functional viral and plasmid DNAvectors capable of expression and replication in a host are known in theart. Such vectors are used to incorporate DNA sequences of theinvention.

DNA sequences encoding the polypeptides can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, in other wordswhen the foreign DNA is continuously maintained in the host, are knownin the art.

In the present invention, the JNK polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of thegenetic sequences. Such expression vectors contain a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence of the host. The expression vector typically contains an originof replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg et al., Gene56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

The vector may include a phenotypically selectable marker to identifyhost cells which contain the expression vector. Examples of markerstypically used in prokaryotic expression vectors include antibioticresistance genes for ampicillin (β-lactamases), tetracycline andchloramphenicol (chloramphenicol acetyl-transferase). Examples of suchmarkers typically used in mammalian expression vectors include the genefor adenosine deaminase. (ADA), aminoglycoside phosphotransferase (neo,G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase(HPH), thymidine kinase (TK), and xanthine guaninephosphoribosyltransferse (XGPRT, gpt).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques which are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method by procedures well known in the art. Alternatively, MgCl₂ or RbClcan be used. Transformation can also be performed after forming aprotoplast of the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the polypeptides of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982). Examples of mammalianhost cells include COS, BHK, 293, and CHO cells.

Isolation and purification of host cell expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The JNK protein kinase of the invention is useful in a screening methodfor identifying compounds or compositions which affect the activity ofthe kinase. Thus, in another embodiment, the invention provides a methodfor identifying a composition which affects a c-Jun N-terminal kinasecomprising incubating the components, which include the composition tobe tested and the kinase or a polynucleotide encoding the kinase, underconditions sufficient to allow the components to interact, thensubsequently measuring the effect the composition has on kinase activityor on the polynucleotide encoding the kinase. The observed effect on thekinase may be either inhibitory or stimulatory. For example, theincrease or decrease of kinase activity can be measured by adding aradioactive compound to the mixture of components, such as ³²P-ATP, andobserving radioactive incorporation into c-Jun or other suitablesubstrate for JNK, to determine whether the compound inhibits orstimulates protein kinase activity. A polynucleotide encoding the kinasemay be inserted into an expression vector and the effect of acomposition on transcription of the kinase can be measured, for example,by Northern blot analysis.

In another embodiment, the invention provides a method of treating acell proliferative disorder associated with JNK comprising administeringto a subject with the disorder a therapeutically effective amount ofreagent which modulates kinase activity. The term “therapeuticallyeffective” means that the amount of monoclonal antibody or antisensenucleotide, for example, which is used, is of sufficient quantity toameliorate the JNK associated disorder. The term “cell-proliferativedisorder” denotes malignant as well as non-malignant cell populationswhich morphologically often appear to differ from the surroundingtissue. For example, the method may be useful in treating malignanciesof the various organ systems, such as lung, breast, lymphoid,gastrointestinal, and genito-urinary tract as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer, non-small cell carcinoma of the lung, cancerof the small intestine and cancer of the esophagus.

The method is also useful in treating non-malignant orimmunological-related cell-proliferative diseases such as psoriasis,pemphigus vulgaris, Behcet's syndrome, acute respiratory distresssyndrome (ARDS), ischemic heart disease, post-dialysis syndrome,leukemia, rheumatoid arthritis, acquired immune deficiency syndrome,vasculitis, septic shock and other types of acute inflammation, andlipid histiocytosis. Especially preferred are immunopathologicaldisorders. Essentially, any disorder which is etiologically linked toJNK kinase activity would be considered susceptible to treatment.

Treatment includes administration of a reagent which modulates JNKkinase activity. The term “modulate” envisions the suppression ofexpression of JNK when it is over-expressed, or augmentation of JNKexpression when it is under-expressed. It also envisions suppression ofphosphorylation of c-Jun, for example, by using the peptide of SEQ IDNO:1 as a competitive inhibitor of the natural c-Jun binding site in acell. When a cell proliferative disorder is associated with JNKoverexpression, such suppressive reagents as antisense JNKpolynucleotide sequence or JNK binding antibody can be introduced to acell. In addition, an anti-idiotype antibody which binds to a monoclonalantibody which binds a peptide of the invention may also be used in thetherapeutic method of the invention. Alternatively, when a cellproliferative disorder is associated with underexpression or expressionof a mutant JNK polypeptide, a sense polynucleotide sequence (the DNAcoding strand) or JNK polypeptide can be introduced into the cell.

The antibodies of the invention can be administered parenterally byinjection or by gradual infusion over time. The monoclonal antibodies ofthe invention can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration of a peptide or an antibodyof the invention include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like.

Polynucleotide sequences, including antisense sequences, can betherapeutically administered by various techniques known to those ofskill in the art. Such therapy would achieve its therapeutic effect byintroduction of the JNK polynucleotide, into cells of animals having theproliferative disorder. Delivery of JNK polynucleotide can be achievedusing free polynucleotide or a recombinant expression vector such as achimeric virus or a colloidal dispersion system. Especially preferredfor therapeutic delivery of nucleotide sequences is the use of targetedliposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a JNK sequence into the viralvector, along with another gene which encodes the ligand for a receptoron a specific target cell, for example, the vector is now targetspecific. Retroviral vectors can be made target specific by inserting,for example, a polynucleotide encoding a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody totarget the retroviral vector. Those of skill in the art will know of, orcan readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the JNK polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsitation. Helper cell lines which havedeletions of the packaging signal include but are not limited to Ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for JNK polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 um canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genes ofinterest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino, etal., Biotechniques, 6:682, 1988).

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The invention also provides a method for detecting a cell with JNKkinase activity or a cell proliferative disorder associated with JNKcomprising contacting a cell component with c-Jun N-terminal kinaseactivity with a reagent which binds to the component and measuring theinteraction of the reagent with the component. Such reagents can be usedto measure relative levels of JNK expression compared to normal tissue.The cell component can be nucleic acid, such as DNA or RNA, or protein.When the component is nucleic acid, the reagent is a nucleic acid probeor PCR primer. The interaction of a nucleic acid reagent with a nucleicacid encoding a polypeptide with c-Jun N-terminal kinase activity istypically measured using radioactive labels, however, other types oflabels will be known to those of skill in the art. When the cellcomponent is protein, the reagent is typically an antibody probe. Theprobes are directly or indirectly detectably labeled, for example, witha radioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Preferably the probe for identification of a cell with JNK kinaseactivity is a c-Jun protein. JNK activity within a cell is measured bythe amount of phosphorylation of the c-Jun protein probe. For example,the amount of JNK activity in a cell extract can be measured by mixingthe extract with c-Jun protein and adding a radioactive compound such as³²P-ATP to the mixture of components. The amount of radioactivity thatis incorporated into the c-Jun probe is determined, for example bySDS-PAGE, and compared to a cell control containing c-Jun and a normallevel of JNK kinase activity.

The c-Jun substrate can be immobilized onto a 96 well microtiter dishand extracts from treated cells added to the wells. The wells are thenwashed and an appropriate buffer containing ³²P-ATP is added to thewells. The phosphorylation reaction proceeds for about 15 minutes andthe wells are washed and counted. Modifications of the assay includeimmobilizing the substrate using beads or magnetic particles andnon-radioactive procedures to measure the substrate phosphorylation,such as using monoclonal antibodies and a detection system (e.g.,biotinilated antibodies and avidin peroxidase reaction).

The Jun protein used in the method of detection of the JNK kinasedescribed above may exist as a single protein unit or a fusion protein.The fusion protein preferably consists of c-Jun andglutathione-S-transferase (GST) as a carrier protein. The c-junnucleotide sequence is cloned 3′ to the carrier protein in an expressionvector, such as pGEX or such derivatives as pGEX2T or pGEX3X, the geneis expressed, the cells are lysed, and the extract is poured over acolumn containing a resin or mixed directly with a resin to which thecarrier protein binds. When GST is the carrier, a glutathione (GSH)resin is used. When maltose-binding protein (MBP) is the carrier, anamylose resin is used. Other carrier proteins and the appropriatebinding resin will be known to those of skill in the art.

The materials of the invention are ideally suited for the preparation ofa kit. The kit is useful for the detection of the level of a c-JunN-terminal kinase comprising an antibody which binds a c-Jun N-terminalkinase or a nucleic acid probe which hybridizes to JNK nucleotide, thekit comprising a carrier means being compartmentalized to receive inclose confinement therein one or more containers such as vials, tubes,and the like, each of the container means comprising one of the separateelements to be used in the assay. For example, one of the containermeans may comprise a monoclonal antibody of the invention which is, orcan be, detectably labelled. The kit may also have containers containingbuffer(s) and/or a container comprising a reporter-means (for example, abiotin-binding protein, such as avidin or streptavidin) bound to areporter molecule (for example, an enzymatic or fluorescent label).

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Plasmids and Expression of GST Fusion Proteins

The glutathione-S-transferase (GST)-cJun expression vector,pGEX2T-cJun(wt), was constructed by inserting a filled-in BspHI-PstIfragment (encoding AA 1-223) from RSV-cJun(BspHI) into the Smal site ofpGEX2T (Pharmacia). RSV-cJun(BspHI) was constructed by changing thetranslation initiation sequence CTATGA of RSV-cJun to TCATGA bysite-directed mutagenesis. The GSTcJun(Ala63/67)(BspHI) expressionvector was derived in the same manner from RSV-cJun(Ala63/73) (Smeal, etal., supra, 1991) and was used to construct pGEX2T-cJun(Ala 63/67). Thevarious GSTcJun truncation mutants were constructed using the polymerasechain reaction (PCR) to amplify various portions of c-Jun coding region.The sequences of the primers are indicated below:

N-terminal primers: TCTGCAGGATCCCCATGACTGCAAAGATGGAAACG (underlinedcodon: amino acid 1) (SEQ ID NO: 2); TCTGCAGGATCCCCGACGATGCCCTCAACGCCTC(a.a. 11) (SEQ ID NO: 3); TCTGCAGGATCCCCGAGAGCGGACCTTATGGCTAC (a.a. 22)(SEQ ID NO: 4); TCTGCAGGATCCCCGCCGACCCAGTGGGGAGCCTG (a.a. 43) (SEQ IDNO: 5); TCTGCAGGATCCCCAAGAACTCGGACCTCCTCACC (a.a. 56) (SEQ ID NO: 6)C-terminal primers: TGMTTCTGCAGGCGCTCCAGCTCGGGCGA (a.a. 79) (SEQ ID NO:7); and TGAATTCCTGCAGGTCGGCGTGGTGGTGATGTG (a.a. 93) (SEQ ID NO: 8).

The DNA fragments were amplified by using Pfu polymerase (Strategene, LaJolla, Calif.), digested with BamHI and PstI, and subcloned to BamHI,PstI sites of pBluescript SK+ (Strategene). The BamHI-EcoRI fragmentswere excised from pBluescript and subcloned to BamHI, PstI sites ofpGEX3X (Pharmacia). Some constructs were made by inserting BamHI-Avalfragments of the PCR products and the AvaI-EcoRI fragment ofpGEX2T-cJun(wt) into BamHI, EcoRI sites of pGEX3X. pGEX3X-cJun(33-223)was constructed by inserting a XhoII-EcoRI fragment into pGEX3X.

The v-Jun and chick c-Jun sequences were derived from RCAS VC-3 and RCASCJ-3 respectively (Bos, et al., Genes Dev., 4:1677, 1990). GSTfusionvectors for v-Jun and chicken c-Jun were constructed by inserting NcoIfragments of RCAS VC-3 and RCAS CJ-3 into NcoI site of pGEX-KG (Guan andDixon, Anal. Biochem., 192:262, 1989). The same fragments containvarious portions of the c-Jun and v-Jun coding regions were cloned intopSG424, a GAL4 DNA binding domain expression vector (Sadowski andPtashne, Nucl. Acids Res., 17:753, 1989).

The GST fusion protein expression vectors were transformed into theXL1-Blue or NM522 strains of E. coli. Protein induction and purificationwere performed as previously described (Smith and Johnson, Gene, 67:31,1988). The amount of purified fusion protein was estimated by theBio-Rad Protein Assay Kit. In some experiments GST fusion proteins werenot eluted from the glutathione (GSH)-agarose beads and were retained onthe beads for isolation of the c-Jun N-terminal kinase.

Cell Culture and Preparation of Cell Extracts

FR3T3, Ha-ras transformed FR3T3, HeLaS3 and QT6 cells were grown inDulbecco's modified Eagle's Medium (DMEM) containing 10% fetal calfserum (FCS), 100 U/ml penicillin (Pc), and 100 μg/ml streptomycin (Sm).Jurkat, K562 and U937 cells were grown in RPMI 1640 supplemented with10% FCS, 100 U/ml Pc, and 100 μg/ml Sm. F9 cells were grown in 45% DMEM,45% Ham's F12, 10% FCS, 100 U/ml Pc and 100 μg/ml Sm. Nuclear andcytoplasmic extracts were prepared as described by Dignam, et al.,(1983). To prepare whole cell extract (WCE), harvested cells weresuspended in WCE buffer: 25 mM HEPES pH 7.7, 0.3 M NaCl; 1.5 mM MgCl₂0.2mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 20 mM β-glycerophosphate, 0.1 mMN₃VO₄, 2 μg/ml leupeptin, 100 μg/ml PMSF. The cell suspension wasrotated at 4° C. for 30 minutes and the extract was cleared bycentrifugation at 10,000×g for 10 minutes. Protein amount was estimatedby Bio-Rad Protein Assay Kit.

Transfection Experiments

Transfection experiments were performed using RSV-cJun, RSV-vJun andGAL4-Jun, GAL4-vJun and Ha-Ras(Leu 61) expression vectors as previouslydescribed (Boyle, et al., supra, 1991; Binetruy, et al., supra, 1991;Smeal, et al., supra, 1991). CAT activity was determined as described inExample 8 below. c-Jun and v-Jun protein expression and phosphorylationwere analyzed as described by Smeal, et al., supra, 1991; Smeal, et al.,Mol. Cell Biol., 12:3507, 1992).

Protein Purification

GST-fusion proteins were purified by affinity chromatography onGSH-agarose as described (Smith, et al., Gene, 67:31-40, 1988). PurifiedMAP kinase (a mixture of ERK1 and ERK2) was obtained from Dr. M. Cobb(University of Texas Southwestern). JNK-46 was purified fromUV-irradiated HeLa S3 cells by standard liquid chromatography.Epitope-tagged JNK was immunopurified from transiently transfected COScells. Briefly, COS cells were solubilized with 20 mM Tris (pH 7.6),0.5% NP-40, 250 mM NaCl, 3 mM β-glycerophosphate, 3 mM EDTA, 3 mM EGTA,100 μM Na orthovanadate, 10 μg/ml leupeptin, 1 mM PMSF. JNK was isolatedby immunoaffinity chromatography using the M2 monoclonal antibody boundto protein A-Sepharose. The beads were washed extensively with Buffer A(20 mM Hepes (ph 7.7), 50 mM NaCl, 0.1 mM EDTA, 0.05% Triton X-100). JNKwas eluted from the column with 3 M urea in Buffer A and the dialyzedagainst Buffer A with 10% glycerol.

EXAMPLE 2 Kinase Assays

Solid Phase Kinase Assay

Cell extracts were diluted so that the final composition of the WCEbuffer was 20 mM HEPES pH 7.7, 75 mM NaCl, 2.5 mM MgCl₂, 0.1 mM EDTA,0.05% Triton X-100, 0.5 mM DTT, 20 mM β-glycerolphosphate, 0.1 mMNa₃VO₄, 2 μg/ml leupeptin, 100 μg/ml PMSF. The extracts were mixed with10 μl of GSH-agarose suspension (Sigma) to which 10 μg of either GST orGST-Jun fusion proteins were bound. The mixture was rotated at 4° C. for3 hours in a microfuge tube and pelleted by centrifugation at 10,000×gfor 20 sec. After 4×1 ml washes in HEPES binding buffer (20 mM HEPES pH7.7, 50 mM NaCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 0.05% Triton X-100), thepelleted beads were resuspended in 30 μl of kinase buffer (20 mM HEPESpH 7.6, 20 mM MgCl₂, 20 mM β-glycerolphosphate, 20 μM p-nitrophenylphosphate, 0.1 mM Na₃VO₄, 2 mM DTT) containing 20 μM ATP and 5 μCiγ-³²P-ATP. After 20 minutes at 30° C. the reaction was terminated bywashing with HEPES binding buffer. Phosphorylated proteins were elutedwith 30 μl of 1.5×Laemlli sample buffer and resolved on a 10% SDSpolyacrylamide gel, followed by autoradiography. Quantitation ofphosphate incorporated was determined by gel slicing and scintillationcounting. Phosphorylated GST fusion proteins were eluted from gel slicesand subjected to phosphopeptide mapping as described (Boyle, et al.,supra, 1991).

In-Gel Kinase Assay

In-gel kinase assay was performed as described by Kameshita andFujisawa, Anal. Biochem., 183:139, (1989) with slight modifications.Briefly, c-Jun binding proteins were isolated from whole cell extractsby using GSH-agarose beads containing 80 μg GST-cJun as described above.Proteins were eluted in Laemlli sample buffer and resolved on 10%SDS-polyacrylamide gel, which was polymerized in the absence or presenceof GST-cJun (40 μg/ml). After electrophoresis, the gel was washed twice,30 minutes each time with 100 ml of 20% 2-propanol, 50 mM HEPES pH 7.6to remove SDS. After the gel was washed twice, 30 minutes each time,with 100 ml of buffer A (50 mM HEPES pH 7.6, 5 mM β-mercaptoethanol), itwas incubated in 200 ml of 6M urea in buffer A at room temperature for 1hr, followed by serial incubations in buffer A containing 0.05% Tween 20and either 3M, 1.5M or 0.75M urea. After the gel was washed severaltimes, 1 hr each time, with 100 ml of buffer A containing 0.05% Tween 20at 4° C., it was incubated with kinase buffer containing 50 μM ATP and 5μCi/ml γ-³²P-ATP at 30° C. for 1 hour. After the reaction, the gel waswashed with 100 ml of 5% tricholoroacetic acid and 1% sodiumpyrophosphate at room temperature several times, followed by drying andautoradiography.

EXAMPLE 3 Binding of a Protein Kinase to GST-cJun-GSH-AGAROSE Beads

The fusion protein, GSTcJun(wt), can bind through its GST moiety toglutathione (GSH)-agarose beads to generate an affinity matrix foridentification of c-Jun binding proteins, which may include proteinkinases. Ha-ras transformation of FR3T3 cells results in increasedphosphorylation of c-Jun on Ser 63 and 73 (Binetruy, et al., supra,1991; Smeal, et al., supra, 1991). Preliminary experiments indicatedthat transformed cells contained higher levels of c-Jun N-terminalkinase activity, while the levels of c-Jun C-terminal kinase activityremained unchanged. To develop a more convenient assay forcharacterizing the c-Jun N-terminal kinase activity, nuclear andcytoplasmic extracts of untransformed and transformed FR3T3 cells weremixed with GSTcJun(wt)-GSH-agarose beads. FRT3T3(−) andHa-ras-transformed FR3T3(+) cells were kept in 0.5% FCS for 24 hours andharvested to prepare nuclear and cytosolic extracts. These extracts(prepared from equal number of cells) were mixed with GSH-agarose beadscontaining 10 μg of GST-cJun(wt), GSTcJun(Ala63/73) or GST. After a 3hour incubation, the beads were spun down, washed 4-times and incubatedin kinase buffer containing γ-³²P-ATP for 20 minutes at 30° C. Thereaction was terminated by washing in SDS sample buffer. The elutedproteins were resolved by SDS-PAGE. The location of the GSTcJun fusionproteins is indicated in FIG. 1. Similar results were obtained whenprotein concentration rather than cell number (300 μg of cytosolicextract and an equivalent amount of nuclear extract) was used tonormalize the amounts of extracts used in this assay. This procedureresulted in phosphorylation of GSTcJun(wt), suggesting that a proteinkinase bound to it and phosphorylated it while attached to GSH-agarose(FIG. 1). On the other hand, no phosphorylation of GST bound toGSH-agarose could be detected by this assay.

The same experiment was repeated using a GSTcJun(Ala63/73) fusionprotein, in which both the serine at position 63 and 73 were convertedto alanines in order to identify a kinase that targets Ser 63 and 73 ofc-Jun. Phosphorylation of this protein was considerably lower than thatof GSTcJun(wt) (FIG. 1). These experiments confirmed the previousobservations that the kinase activity affecting the N-terminal sites ofc-Jun was elevated upon Has-ras transformation and are consistent withthe differences in the extent of c-Jun N-terminal phosphorylationbetween transformed and untransformed cells detected by in vivolabelling (Binetruy, et al., supra, 1991; Smeal, et al., supra, 1991,1992). The kinase activity detected by this solid-phase assay waspresent in both the cytosolic and the nuclear fractions and wasseveral-fold more abundant in the cytosol on a per-cell basis. However,it is possible that some of the kinase leaked from the nuclei to thecytosol during the cell fractionation.

The solid-phase assay was used to examine N-terminal c-Jun kinaseactivity in other cell types. Exposure of HeLa cells to UV activates theHa-Ras signalling pathway and results in a large increase in N-terminalphosphorylation of c-Jun (Devary, et al., Cell, 71:1081, 1992).Treatment of HeLa cells with the phorbol ester, TPA, on the other hand,has only a marginal effect on N-terminal phosphorylation of c-Jun(Boyle, et al., 1991). HeLa S3 cells were serum starved for 12 hours andwere either left untreated, irradiated with UV light (40 J/m²) orincubated with TPA (100 ng/ml). The cells were harvested at theindicated times (min) after UV or TPA exposure. Whole cell extracts(approximately 800 μg protein) isolated form equal numbers of cells weremixed with GSH-agarose beads containing 10 μg of either GST,GSTcJun(wt), or GSTcJun(Ala 63/73). After 3 hours incubation, followedby extensive washing, the solid state phosphorylation assay wasperformed as described above. After a 20 minute reaction, the proteinswere dissociated in SDS sample buffer and resolved by SDS-PAGE.

As shown in FIG. 2A, N-terminal c-Jun kinase activity was elevatedwithin 5 minutes after UV irradiation and was 250-fold higher after 30minutes than in unstimulated cells. The effect of TPA, however, wasminor compared to that of UV. As found before, GSTcJun(wt) was moreefficiently phosphorylated than GSTcJun(Ala63/73), whereas GST was notphosphorylated. These results are consistent with in vivo measurementsof c-Jun phosphorylation (Boyle, et al., supra, 1991; Devary, et al.,supra, 1992).

TPA treatment of Jurkat T cells, in contrast to HeLa cells, resulted instimulation of c-Jun phosphorylation on Ser 63 and 73. Jurkat cells wereserum starved for 2 hours and either left untreated or stimulated withTPA (50 ng/ml) for 10 or 30 minutes. Whole cell extracts prepared from5×10⁶ cells were mixed with GSH-agarose beads containing GST,GSTcJun(wt) or GSTcJun(Ala63/73). Phosphorylation of the GST proteinsattached to the beads was performed as described above. The fastermoving bands correspond to degradation products of the GSTcJun proteins.

In Jurkat cells, unlike HeLa cells, the N-terminal kinase activity wasfound to be strongly activated by TPA (25-fold after 30 minutes) (FIG.2B). This kinase also preferred GSTcJun(wt) over GSTcJun(Ala63/73) anddid not bind to or phosphorylate the GST moiety. Collectively, thesefindings suggest that the kinase detected by the solid-phase assayphosphorylates c-Jun on Ser 63 and 73 and that its regulation parallelsthat of c-Jun N-terminal phosphorylation examined by in vivo labelling.

EXAMPLE 4 Phosphorylation of Serines 63 and 73 by Bound Kinase, JNK

To determine the exact phosphoacceptor sites used by the kinase thatbinds to GSTcJun, the phosphorylated GSTcJun(wt) and GSTcJun(Ala63/73)proteins were subjected to two-dimensional tryptic phosphopeptidemapping. Whole cell extracts of Ha-ras-transformed FR3T3 cells (2.5 mg),UV irradiated HeLa cells (200 μg) or TPA-stimulated Jurkat cells (1.2mg) were mixed with GSH-agarose beads, containing either GSTcJun(wt) orGSTcJun(Ala63/73). The GSTcJun proteins were phosphorylated as describedabove by the bound kinase, isolated by SDS-PAGE, excised from the gel,digested with trypsin and subjected to two-dimensional phosphopeptidemapping. The X, Y, T1, and T2 phosphopeptides are indicated. All theautoradiograms were exposed for the same length of time.

As shown in FIG. 3A, the kinases isolated from Ha-ras-transformed FR3T3cells, UV-irradiated HeLa cells and TPA-stimulated Jurkat cells,phosphorylated GSTcJun on X, Y, and two other peptides, T1 and T2 X andY correspond to phosphorylation of Ser-73 and Ser-63, respectively(Smeal, et al., supra, 1991) and were absent in digests ofGSTcJun(A1a63/73), which contained higher relative levels of T1 and T2.Phosphoaminoacid analysis indicated that T1 and T2 contain onlyphosphothreonine. By deletion analysis these threonines were assigned toAA 91, 93 or 95 of c-Jun.

As described below, the kinase bound to GSTcJun was eluted from thebeads and used to phosphorylate recombinant full-length c-Jun protein insolution (FIG. 3B). Recombinant c-Jun protein was phosphorylated invitro by the c-Jun N-terminal kinase (JNK) eluted fromGSTcJun(WT)-GSH-agarose beads. In addition, c-Jun was isolated byimmuneprecipitation from ³²P-labelled F9 cells that were cotransfectedwith c-Jun and Ha-Ras expression vectors (Smeal, et al., supra, 1991).Equal counts of each protein preparation were digested with trypsin andsubjected to phosphopeptide mapping. The migration positions of the X,X′ (a derivative of X generated by alkylation; Smeal, et al., supra,1991) Y, b and c phosphopeptides are indicated.

As found in vivo, the bound kinase phosphorylated c-Jun mostly on Ser73, followed by phosphorylation of Ser 63. In addition, the bound kinaseactivity phosphorylated c-Jun weakly on two of its C-terminal sites,resulting in appearance of phosphopeptides b and c. Since this is thefirst protein kinase that was detected with clear specificity for atleast one of the N-terminal sites of c-Jun, it was named JNK, for cJunN-terminal protein-kinase.

EXAMPLE 5 Binding of JNK to cJun

To examine the stability of the interaction between GSTcjun and JNK,extracts of TPA-stimulated Jurkat cells were incubated withGSTcJun(wt)-GSH-agarose beads. After extensive washing, the beads weresubjected to elution with increasing concentrations of NaCl, urea,guanidine-HCl and SDS. Elution of JNK was examined by its ability tophosphorylate recombinant c-Jun in solution. GSTcjun(wt)-GSH-agarosebeads were incubated for 3 hours with a whole cell extract ofTPA-stimulated Jurkat cells and after four washes were subjected toelution in kinase buffer containing increasing concentrations of NaCl,urea, guanidine-HCl (in M) or SDS (in %)(FIG. 4). The eluted fractions(equal volumes) were dialyzed at 4° C. against kinase buffer containing10% glycerol and no ATP and then incubated with recombinant c-Junprotein (250 ng) in the presence of 20 μM ATP and 5 μCi of γ-³²P-ATP for20 minutes at 30° C. The amount of kinase remaining on the beads afterthe elution steps (R lanes) was determined by incubation of the isolatedbeads with kinase buffer in the presence of 20 μM ATP and 5 μCi γ-P-ATPfor 20 minutes at 30° C. The phosphorylated proteins were analyzed bySDS-PAGE as described above and visualized by autoradiography. Themigration positions of GSTcJun and c-Jun are indicated.

Surprisingly, JNK was found to bind GSTcJun rather tightly; only a smallfraction of kinase activity was eluted by 0.5M NaCl and even afterelution with 2M NaCl, most of the kinase remained on the beads (FIG.4A). Approximately 50% of the bound kinase was eluted by 1M urea and therest was eluted by 2M urea. Nearly complete elution was achieved byeither 0.5M guanidine-HCl or 0.01% SDS. Under all of these elutionconditions, GSTcJun(wt) was also partially eluted from the GSH-agarosebeads. This suggests that the stability of the JNK:c-Jun complex issimilar to that of the GST:GSH complex.

GSTcJun(wt) was covalently linked to GSH-agarose beads, usingcyanogenbromide, and incubated with a whole cell extract ofTPA-stimulated Jurkat cells. After extensive washing, part of the beadswere eluted with kinase buffer-containing: no ATP (FIG. 4B, lane 2), 20μM ATP (lane 3) or 50 μM ATP (lane 4). The eluted fractions (equalvolumes) were incubated with recombinant c-Jun protein (500 ng) as asubstrate and 5 μCi γ-³²P-ATP for 30 minutes. In addition, the beadsafter elution with either kinase buffer alone (lane 1) or kinase buffercontaining 50 μM ATP (lane 5) were incubated with c-Jun protein (500 ng)in the presence of 5 μCi γ-³²P-ATP for 30 minutes. Phosphorylation ofc-Jun (indicted by the arrow) was analyzed by SDS-PAGE andautoradiography.

Addition of exogenous c-Jun to kinase-loaded GSH-agarose beads to whichGSTcJun was covalently linked results in its efficient phosphorylation(FIG. 4B, Lane 1). This suggests that after phosphorylating GSTcJun, JNKdissociates from it and phosphorylates exogenous c-Jun. In addition,incubation with kinase buffer containing ATP resulted in elution of JNKfrom the GSTcJun beads, as indicated by its ability to phosphorylateexogenous c-Jun (FIG. 4B, lanes 2-4). After incubation with 50 μM ATPless than 20% of the kinase remained on the beads (compare lanes 1 and5, FIG. 4B).

EXAMPLE 6 JNK1 is a 46 kD Protein

An in-gel kinase assay was performed to determine the size of JNK.GSTcJun-GSH-agarose beads were incubated with a whole cell extract ofTPA-stimulated Jurkat cells, washed extensively and the bound proteinswere eluted in SDS sample buffer and separated on SDS-polyacrylamidegels that were polymerized in the absence (−) or presence (+) ofGSTcJun(wt). After electrophoresis, the gel was incubated in 6 M ureaand subjected to renaturation as described in Example 1. The renaturedgels were incubated in kinase buffer containing 50 μM ATP and 5 μCi/mlγ-³²P-ATP for 1 hour at 30° C., washed, fixed, and visualized byautoradiography.

In both cases a protein band whose apparent molecular weight was 46 kDwas phosphorylated (FIG. 5A). Phosphorylation was 2-fold more efficientin the presence of GSTcJun. This indicates that 46 kD protein band iseither autophosphorylated JNK or a comigrating protein. No ³²P-labelledprotein was detected in eluates of GST-GSH-agarose beads.

The same in-gel kinase assay was used to demonstrate increased JNKactivity upon TPA stimulation of Jurkat cells or UV irradiation of HeLacells (FIG. 5B). GSTcJun-GSH-agarose beads were incubated with wholecell extracts of unstimulated or UV-stimulated HeLa cells andunstimulated or TPA-stimulated Jurkat cells. After washing, the boundproteins were eluted in SDS sample buffer and separated by SDS-PAGE.After renaturation, the gel was incubated in kinase buffer containing 50μM ATP and 5 μCi/ml γ-³²P-ATP and the phosphorylated proteins werevisualized by autoradiography.

These results provide further evidence that the apparent molecularweight of JNK is 46 kD. To determine whether the same N-terminal c-Junkinase is present in various cell types, the in-gel kinase assay wasused to examine extracts of K562 human erythroleukemia cells, U937 humanhistiocytic leukemia cells, Jurkat cells, HeLa cells, F9 embryonalcarcinoma cells, Ha-ras-trans-formed FR3T3 cells and QT6 quailfibroblasts. The HeLa, F9 and QT6 extracts were prepared formUV-irradiated cells and the U937 and Jurkat extracts were made fromTPA-stimulated cells, while the K562 cells were not subjected to anyspecial treatment. All cells contained a protein kinase that bound toGSTcJun and migrated around 46 kD (FIG. 5C). Some cells, especially QT6cells, contained a second less abundant protein kinase species,migrating at about 55 kD. The activities of both kinases were induced bycell stimulation. GSTcJun(WT)-GSH-agarose beads were incubated withwhole cell extracts of logarithmically growing K562 and Ha-rastransformed FR3T3 cells, TPA-stimulated Jurkat and U937 cells andUV-irradiated HeLa, F9 and QT6 cells. After washing, the bound proteinswere eluted and analyzed by in-gel kinase assay as described above.

Further evidence that JNK is 46 kD in size was obtained by separatingthe GSTcJun-bound protein fraction of TPA-stimulated Jurkat cell extractby SDS-PAGE. After elution and renaturation of the fractionatedproteins, the molecular weight of the major protein kinase bound toGSTcJun, capable of specific phosphorylation of Ser 63 and 73, wasdetermined to be 46 kD. Although the sizes of ERK1 and ERK2, 44 and 42kD, respectively, are close to that of JNK, Western blot analysis, usingan antiserum that reacts with both ERK's, indicates that the 46 kD JNKis not immunologically related to either of them. In addition, JNK isnot immunologically related to Raf-1. In addition, a 55 kD polypeptidewas identified as exhibiting JNK activity, however, the 46 kD appears tobind c-Jun more efficiently (Hibi, et al., Genes Dev., 7:2135, 1993).

EXAMPLE 7 Delineation of the Kinase Binding Site

Deletion mutants of GSTcJun lacking either N-terminal or C-terminalsequences (FIG. 6A) were used to define the JNK binding site. GSTcJunfusion proteins containing various c-Jun sequences were expressed in E.coli and isolated by binding to GSH-agarose. The bound proteins wereanalyzed by SDS-PAGE and stained with Coomassie Blue. Numbers indicatethe amino acids of c-Jun present in each fusion protein. The migrationpositions of the intact GST-fusion proteins are indicated by the dots.Faster migrating bands are degradation products.

These proteins were immobilized on GSH-agarose beads and incubated withan extract of UV-irradiated HeLa cells. Whole cell extracts ofUV-irradiated HeLa S3 cells were mixed with GSH-agarose beads containingequal amounts of the various GST fusion proteins. After washing, thebeads were incubated for 20 minutes in kinase buffer containingγ-³²P-ATP. The GST-fusion proteins were eluted from the beads andanalyzed by SDS-PAGE and autoradiography. The migration positions of theintact GST fusion proteins are indicated by the dots. After incubationwith whole cell extracts of UV-irradiated HeLa cells and washing, partof the bound JNK fraction was eluted with 1 M NaCl and examined for itsability to phosphorylate recombinant c-Jun (250 ng) in solution. Proteinphosphbrylation was analyzed by SDS-PAGE and autoradiography.

Binding of JNK was examined by its ability to phosphorylate the GSTcJunfusion proteins, all of which contained both Ser 63 and 73 (FIG. 6B). Toexclude the possibility that any of the truncations may have altered theconformation of c-Jun affecting the presentation of its N-terminalphosphoacceptors without affecting JNK binding, the kinase eluted fromthese beads was examined for its ability to phosphorylate exogenousfull-length c-Jun in solution (FIG. 6C). The results obtained by bothassays indicated that removal of amino acids (AA) 1-21 had no effect onJNK binding. Removal of AA 1-32 decreased phosphorylation of GSTcJun buthad only a small effect on kinase binding. Removal of AA 1-42, however,completely eliminated kinase binding. In contrast to the N-terminaltruncations, the two. C-terminal truncations, that were examined, had noeffect on JNK binding and a GST fusion protein containing AA 1-79 ofc-Jun exhibited full binding activity. Hence, AA 33-79 constitute thekinase binding site.

The JNK binding site encompasses the δ region, spanning AA 31-57 ofc-Jun that are deleted in v-Jun (Vogt and Bos, 1990). To determine theinvolvement of the δ region in kinase binding, GST fusion proteinscontaining the N-terminal activation domain of chicken c-Jun (AA 1-144),or the equivalent region of v-Jun (FIG. 7A) were constructed. Theactivation domain (AA 1-144) of chicken (ch) c-Jun and the equivalentregion of v-Jun were fused to GST and expressed in E. coli. GST fusionproteins were isolated on GSH-agarose beads and analyzed by SDS-PAGE andCoomassie Blue staining. The migration positions of the intact proteinsare indicated by the dots. After loading these GST fusion proteins ontoGSH-agarose the kinase binding assays were performed as described above.

Extracts of TPA-activated Jurkat cells were incubated with GSH-agarosebeads containing GST, GSTcJun(Ch) or GSTvJun. After washing, the beadswere incubated in kinase buffer containing γ-³²P-ATP and thephosphorylated GST fusion protein were analyzed as described for FIG. 6.The bound protein fraction was eluted from the GSTcJun(Ch) and GSTvJunbeads and analyzed for its ability to phosphorylate c-Jun in solution,as described for FIG. 6. While chicken GSTcJun bound the kinase asefficiently as human GSTcJun, GSTvJun was defective in JNK binding(FIGS. 7B, C).

EXAMPLE 8 JNK Binding is Required for Ha-Ras and UV Responsiveness

Phosphorylation of Ser 63 and 73 is necessary for potentiation of c-Junmediated transactivation by Ha-Ras (Smeal, et al., supra, 1991). Ifbinding of JNK has any role in this response, mutations that decreasekinase binding in vitro should attenuate the stimulation of c-Junactivity by Ha-Ras in vivo. This relationship was examined bycotransfection assays. Expression vectors were constructed to expresschimeric GAL4-cJun and GAL4-vJun proteins, that consist of the DNAbinding domain of the yeast activator GAL4 (Sadowski and Ptashne, 1989)and N-terminal sequences of c-Jun or v-Jun. The ability of thesechimeras to activate the GAL4-dependent reporter 5×GAL4-Elb-CAT (Lillieand Green, 1989) was examined in the absence or presence of acotransfected Ha-Ras expression vector (FIG. 8A). F9 cells werecotransfected with 1.0 μg of expression vector encoding the indicatedGAL4-cJun chimeric proteins containing various portions of the c-Junactivation domain [cJ=AA1-223; 33-AA33-223; 56=AA56-223; A63,73=AA1-246(Ala63/73)] and 2.0 μg of a 5×GAL4-Elb-CAT reporter in theabsence or presence of the indicated amounts (in μg) ofpZIPNeoRas(Leu61). The total amount of expression vector was keptconstant and the total amount of transfected DNA was brought to 15 μgusing pUC18 and the appropriate amount of pZIPneo. Cells were harvested28 hours after transfection and CAT activity was determined. Shown arethe averages of two experiments, calculated as fold-activation over thelevel of reporter expression seen in the absence of the GAL4-Junexpressions vectors.

While deletion of AA 1-32 of c-Jun resulted in a small decrease inHa-Ras responsiveness (9.8-fold induction vs. 19-fold induction for wtGAL4-cJun), deletion of AA 1-42 or 1-55 resulted in a greater decreasein Ha-Ras responsiveness (5.2-fold induction). A similar decrease inHa-Ras responsiveness was observed upon substitution of c-Jun sequenceswith v-Jun sequences (4.7-fold induction). In fact, theGAL4-cJun(56-223) and GAL4-vJun chimeras were only 2-fold moreresponsive than GAL4-cJun(1-246;Ala63/73) in which Ser 63 and 73 wereconverted to alanines. That chimera exhibited only a marginal response(2-fold) to Ha-Ras. The same set of GAL4-cJun and GAL4-vJun fusionproteins was tested for UV responsiveness. F9 cells were transfected asdescribed above except that instead of cotransfection with pZIPNeoRas,the cells were either exposed or not exposed to 40 J/m2 of UV-C 8 hoursafter transfection. The cells were harvested and assayed for CATactivity 20 hours later. FIG. 8B shows the averages of two experimentscalculated as described above.

As shown in FIG. 8B, those proteins incapable of binding JNK in vitro,were non-responsive to UV in vivo. While the activity ofGAL4-cJun(43-223) was stimulated 7.5-fold by UV, the activities ofGAL4-cJun(43-233), GAL4-cJun(56-223) and GAL4-vJun were induced only1.5-fold.

To reveal the role of JNK binding in c-Jun phosphorylation, F9 cellswere transfected with c-Jun and v-Jun expression vectors in the absenceor presence of an activated Ha-Ras expression vector. UV-irradiation wasalso used to activate the Ha-Ras pathway (Devary, et al., 1992). v-Junand c-Jun were isolated by immunoprecipitation from ³²S- or ³²P-labelledF9 cells that were transfected with v-Jun and c-Jun expression vectorsin the absence or presence of pZIPNeoRas (Leu61). The isolated proteinswere analyzed by SDS-PAGE and autoradiography. Shown are the results ofone typical experiment for each protein. Note that the ³²P-labelledv-Jun autoradiogram was exposed 3 times longer than the correspondingc-Jun autoradiogram to generate signals of similar intensity. v-Jun andc-Jun were isolated from ³²P and ³²S-labelled F9 cells that weretransfected with v-Jun or c-Jun expression vectors. One half of thecells were irradiated with UV-C(40 J/m²) for 30 minutes prior toisolation of the Jun proteins by immunoprecipitation. In this case, thec-Jun and v-Jun lanes represent equal autoradiographic exposures. Thetwo arrowheads indicate the migration positions of the two forms ofc-Jun (Devary, et al., 1992), whereas the square indicates the migrationposition of v-Jun.

Immunoprecipitation from ³²S-labelled cells showed that c-Jun and v-Junwere expressed at similar levels and that their expression level was notaffected by either Ha-Ras (FIG. 9A) or UV (FIG. 9B). Immunoprecipitationfrom ³²P-labeled cells indicated that both Ha-Ras and UV stimulated thephosphorylation of c-Jun, whereas the phosphbrylation of v-Jun, whosebasal level was several-fold lower than that of c-Jun, was not enhancedby either treatment. As observed previously (Devary, et al., supra,1991), UV was a stronger inducer of c-Jun phosphorylation resulting inits retarded electrophoretic mobility. Phosphopeptide mapping confirmedthat Ha-Ras expression had a much smaller effect on the phosphorylationof v-Jun in comparison to its effect on c-Jun. As shown previously(Smeal, et al., supra, 1991), v-Jun was phosphorylated only on one sitewhich is equivalent to Ser 73 of c-Jun.

EXAMPLE 9

Antisera and Proteins

c-Jun polyclonal antiserum was described by Binetruy, et al., (Nature,351:122—127, 1991). The anti-CD3 monoclonal antibody OKT3 (Van Wauwe, etal., J. Immunol., 124:2708-2713, 1980) was obtained from Dr. AmnonAltman, La Jolla Institute for Allergy and Immunology, and the anti-CD28monoclonal antibody 9.3 is described in Hansen, et al., (Immunogenetics,10:247-260, 1980). The anti-ERK2 and anti-ERK antibodies were providedby Drs. M. Weber and M. Cobb (University of Texas Southwestern),respectively. Expression and purification of GST-cJun(1-223) wasdescribed (Hibi, et al., Genes & Dev., 7:2135, 1993). The bacterialexpression vector for kinase-defective ERK-1 was a gift from Dr. M. Cobband the recombinant protein was prepared and purified by Dr. J.Hagstrom. MBP was purchased from Sigma.

Cell Culture, Metabolic Labeling, and Immunoprecipitation

Jurkat cells were grown in RPMI with 10% fetal calf serum (FCS), 1 mMglutamate, 100 μ/ml penicillin (pen), 100 μg/ml streptomycin (strep) and250 ng/ml amphotericin (complete medium). HeLa S3, CV-1 and FR3T3 cellswere grown in DMEM supplemented with 10% FCS, 100 μ/ml pen, 100 μg/mlstrep. All cells were cultured at 37° C. with 5% CO₂. Mouse thymocyteswere prepared from 8 week old Balb/C mice by gradient centrifugation onlymphocyte separation medium (Pharmacia). The lymphocytes were culturedfor 5 hours at 37° C. in RPMI+10% FCS, prior to stimulation. Jurkatcells were labelled for 90 minutes with 0.5 mCi/ml ³²P-orthophosphate(ICN Radiochemicals) in medium lacking sodium phosphate. Labelled cellswere treated with TPA (Sigma) and A23187 (Calbiochem) 1 μg/ml asindicated. When used, cyclosporin A (CsA) (Sandoz) 100 ng/ml in ethanolwas added 10 minutes prior to cell stimulation. Following stimulation,the labelled cells were washed twice with ice-cold PBS then lysed withRIPA buffer (10 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton-X 100,1% DOC, 0.1% SDS) supplemented with phosphatase inhibitors (20 mMβ-glycerophosphate, 10 mM p-nitro-phenylphosphate, 1 mM Na₃ VO₄), andprotease inhibitors (10 μg/ml leupeptin, aprotonin, pepstatin and 1 mMphenylmethyl sulfonylfluoride). c-Jun was immunoprecipitated asdescribed (Binetruy, et al., supra., 1992) and analyzed by SDS-PAGE,followed by peptide mapping (Boyle, et al., Cell, 64:573-584, 1991; Lin,et al. Cell, 70:777-789, 1992). Ha-Ras was immunoprecipitated withY13-259. Ha-Ras bound nucleotides were extracted and analyzed asdescribed by Satoh, et al., (Proc. Natl. Acad. Sci., USA, 15:5993-5997,1990).

RNA Extraction and Northern Blot Analysis

Exponentially growing Jurkat cells (10⁶/ml) grown in complete RPMImedium was pretreated with CsA for 15 minutes when applicable, thensubjected to various treatments for another 40 minutes. Totalcytoplasmic RNA was extracted as previously described (Angel, et al.,Cell, 49:729-739, 1987). 10 μg RNA was denatured by incubating withglyoxal for 60 minutes at 55° C. and fractionated on a 1% agarose gel inphosphate buffer. The fractionated RNA was blotted to Zetabind Nylonmembrane (CUNO Labs) and hybridized to ³²P-labelled cDNA probes specificfor c-jun, jun-B, jun-D, c-fos, α-tubulin and IL-2.

Protein Kinase Assays

Exponentially growing cells were stimulated for the indicated times andhypotonic detergent cellular extracts were prepared as described (Hibi,et al., Genes and Dev., supra, 1993). The solid-state phosphorylationassay for measuring JNK activity was performed by incubated extractswith GSTcJun(1-223)-GSH agarose beads as described (Hibi, et al.,supra., 1993) and as in Example 2. ERK1 and 2 activity was assayed by animmunecomplex kinase assay using MBP as a substrate (Minden, et al.,Nature, 1993).

Reporters, Expression Vectors and Transfections

−79 jun-LUC, −73/+63 Col-LUC, −60/+63 Col-LUC were described previously(Deng and Karin, 1993). The IL2-LUC reporter plasmid was constructed bysubcloning the IL-2 promoter (298 bp) from IL2CAT/+1 (Serfling, et al.,EMBO J., 8:465-473, 1988) into the p20Luc vector (Deng and Karin, Genesand Dev., 7:479, 1993) between the SacI and KpnI site. The c-Junexpression vector pSRaIIc-Jun was constructed by subcloning the humanc-jun HindIII-NotI fragment from pRSVc-Jun (Binetruy, et al., supra.,1991) into pSRαII vector by blunt end ligation. pBJ-CNA and pBJ-CNB werefrom Dr. G. Crabtree, Stanford University. β-Actin-LUC was from Dr. C.Glass, UCSD.

T Ag Jurkat cells, a derivative of the human Jurkat T-cell line stablytransfected with the SV40 large T antigen (a gift from Dr. G. Crabtree)were grown to 10⁶/ml, then resuspended at 2×10⁷/ml in fresh completemedium. 10⁷ cells (0.5 ml) were mixed with reporter plasmids (5 μg, −79jun-LUC; 10 μg, −73/+63 Col-LUC or −60/+63 Col-LUC; 5 μg IL2-LUC) atroom temperature for 10 minutes, then electroporated at 250 V, 960 uF ina 0.4 cm cuvette using a Bio-Rad GenePulser. After electroporation,cells were immediately put on ice for 10 minutes, then resuspended in 10ml complete medium for 24 hours before stimulation. 0.5 μg ofpSRaIIc-Jun were used to transfect 10⁷ Jurkat cells. Luciferase activitywas determined as described (Deng and Karin, supra., 1993).

Analysis of GDP and GTP Bound to RAS p21

Jurkat cells 10×10⁶ were labelled for 3 hours with ³²P-orthophosphate(ICN Radiochemicals) at 1 mCi/ml in 5 mM of Na₃VO₄ phosphate-free DMEMsupplemented with 1 mg/ml BSA. Before harvest, cells were stimulatedwith TPA, 10 ng/ml, A23187, 1 μg/ml anti-CD3 antibody (OKT3), 10 μg/ml,anti-CD28 antibody, 2 μg/ml or their combinations. After treatment for aspecified period, cells were washed once immediately with ice cold PBS,twice with ice-cold Tris-Buffered saline (50 mM Tris-HCl, pH 7.5, 20 mMMgCl₂, 150 mM NaCl, 10.5% Nonidet P-40/1 μg/ml of aprotinin, leupeptin,pepstatin and 1 mM phenylmethyl sulfonylfluoride). Ras p21 wasimmunoprecipitated with monoclonal antibody Y 12-259 (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). The GDP/GTP content of Ras wasanalyzed by TLC as described (Satoh, et al., Proc. Natl. Acad. Sci.,USA, 15:5993, 1990) and quantitated with an Ambis radioanalytic imagesystem (Ambis, San Diego, Calif.).

EXAMPLE 10 Synergistic Induction of AP-1 Activity During T CellActivation

During the first stage of T lymphocyte activation, early response genesare rapidly included (Crabtree, Science, 243:355,361, 1989; Zipfel, etal., Mol Cell Bio., 9:1041-1048, 1989). Induction of jun and fos genesduring activation of the Jurkat T cell line was investigated. Twodifferent co-stimulatory paradigms were used, one employing TPA and theCa²⁺ ionophore A23187, and the second based on simultaneous stimulationof the TCR complex with an antibody to its CD3 component (OKT3; VanWauwe, et al., J. Immunol., 124:2708-2713, 1980) and stimulation of theCD28 auxiliary receptor with an anti-CD28 antibody (9.3; Hansen, et al.,Immunogenetics, 10:247-260, 1993; June, et al., Immunol. Today,11:211-216, 1990). Total cytoplasmic RNA was extracted from Jurkat cellsthat were incubated with 50 ng/ml TPA (T), 1 μg/ml A23187 (A) or 100ng/ml cyclosporin A (CsA) for 40 minutes, either alone or incombinations, as indicated. After fractionation of 10 μg samples on anagarose gel and transfer to nylon membrane, the level of c-jun, jun-B,jun-D, c-fos and α-tubulin expression was determined by hybridization torandom primed cDNA probes.

Second, Jurkat cells were incubated with 10 μg/ml soluble anti-CD3(OKT3), 2 μg/ml soluble anti-CD28 (9.3) or a combination of 50 ng/ml TPAand 1 μg/ml A23817 (T/A) as indicated for 40 minutes. Total cytoplasmicRNA was isolated and 10 μg samples were analyzed as described aboveusing c-jun, jun-D and c-fos probes. IL-2 induction by the sametreatments was measured after 6 hours of stimulation by blothybridization using IL-2 and α-tubulin specific probes.

Both the first and second costimulatory paradigms induced IL-2transcription (FIG. 11B). Optimal induction of c-jun also required acombined treatment with TPA and A23187 (FIG. 11A) or anti-CD3 andanti-CD28 (FIG. 11B). The synergistic induction of c-jun by bothcostimulatory paradigms was partially inhibited by CsA. jun-B was alsoinduced by TPA, but its induction was not affected by A23187 or CsA.Although TPA+A23187 potentiated jun-D expression, this effect was alsonot inhibited by CsA. As reported by Matilla, et al., (EMBO J., 9:4425-4433, 1990), maximal induction of c-fos also required treatmentwith TPA+A23187, but was not inhibited by CsA. Therefore sensitivity toCsA is unique to c-jun. While incubation with soluble anti-CD3 led toinduction of c-jun and c-fos, only c-jun expression was augmented bysimultaneous exposure to anti-CD28.

The effects of the different stimuli on AP-1 transcriptional activity inJurkat cells were examined using a truncated, AP-1 responsive, humancollagenase promoter (Angel, et al., Cell, 49:729-739, 1987) fused tothe luciferase (LUC) reporter gene. Jurkat cells were transfected with10 μg of either −73Col-LUC or −60Col-LUC reporter plasmids. 24 hoursafter transfection, the cells were aliquoted into 24 well plates andincubated for 9 hours with 50 ng/ml TPA, 1 μg/ml A23187 or 100 ng/mlCsA, either alone or in combinations, as indicated. The cells wereharvested and luciferase activity was determined. The results shown areaverages of three experiments done in triplicates.

While TPA and A23187 administered alone had marginal effects on−73Col-LUC, the two together resulted in its synergistic activation(FIG. 11C). The −60Col-LUC reporter, lacking an AP-1 binding site, wasnot induced. Induction of −73Col-LUC was inhibited by CsA. Treatmentwith anti-CD3 and anti-CD28 also resulted in synergistic activation of−73Col-LUC. Similar results were obtained with the AP-1 responsive c-junpromoter. These findings differ from previous measurements of AP-1activity in Jurkat cells that relied on the use of synthetic promoterscontaining multiple AP-1 sites (Matilla, et al., supra, 1993; Ullman, etal., Genes & Dev., 7:188-196, 1993). While these findings werereproducible, previous studies indicate that the physiologicalcollagenase and c-jun promoters provide a more accurate and validmeasurement of AP-1 transcriptional activity. Indeed, the expressionpatterns of the collagenase and c-jun reporters are very similar to thatof the c-jun gene.

EXAMPLE 11 Costimulation of c-Jun N-terminal Phosphorylation isSuppressed by CsA

Induction of c-Jun transcription and optimal stimulation of AP-1correlate with changes in c-jun phosphorylation (Devary, et al., Cell,71:1081-1091, 1992). The effect of TPA and A23187 on c-Junphosphorylation in Jurkat cells was examined. To elevate c-Junexpression, Jurkat cells were transfected with a c-Jun expressionvector. The cells were labelled with ³²P and c-Jun wasimmunoprecipitated from cells subjected to various stimuli and analyzedby SDS-PAGE (FIG. 12A). Jurkat cells (10⁶ cells per lane) weretransfected with 0.5 μg of a SRα-cJun expression vector and 24 hourslater were labeled for 3 hours with ³²P-orthophosphate (1 mCi/ml). After15 minutes, treatment with 50 ng/ml TPA (T), 1 μg/ml A23187 (A) and 100ng/ml CsA, either alone or in combination, as indicated, the cells werelysed in RIPA buffer and c-Jun was isolated by immunoprecipitation andanalyzed by SDS-PAGE. The c-Jun bands are indicated.

In unstimulated cells, phosphorylated c-Jun migrated as a single band.Treatment with TPA for 15 minutes induced the appearance of slowermigrating bands and costimulation with A23187 enhanced this effect,while CsA reduced the Ca⁺⁺ effect. Within the short time frame of thisexperiment, there were minimal effects on c-Jun expression.

Similar results were obtained by analysis of endogenous c-Jun expressionand phosphorylation (FIG. 12B). 2×10⁷ Jurkat cells were labeled for 3hours with either ³⁵S-methionine (900 μCi/ml) or ³²P-orthophosphate (1mCi/ml). After 15 minutes incubation with 50 ng/ml TPA+1 μg/ml A23178(T/A) in the absence or presence of and 100 ng/ml CsA or no addition, asindicated, the cells were lysed in RIPA buffer and c-Jun isolated byimmunoprecipitation and analyzed by SDS-PAGE. The c-Jun band isindicated. However, due to lower expression levels, some of the slowermigrating forms were not clearly visible.

c-Jun phosphorylation was further analyzed by two-dimensionalphosphopeptide mapping (FIG. 12C). This analysis included all theisoforms of c-Jun. All of the c-Jun specific protein bands shown in FIG.12A, isolated from equal numbers of cells, were excised from the gel andsubjected to tryptic phosphopeptide mapping. Shown is a typical result(this experiment was repeated at least three times). N-nonstimulatedcells; T-cells treated with 50 ng/ml TPA; T/A: cells treated with 50ng/ml TPA and 1 μg/ml A23187; T/A+CsA: cells treated with T/A and 100ng/ml CsA. a,b,c,x and y correspond to the various trypticphosphopeptides of c-Jun, previously described by Boyle, et al., (Cell,64:573-584, 1991) and Smeal, et al., (Nature, 354:494-496, 1991). T1 andT2 correspond to the minor phosphorylation sites; Thr91, 93 and 95(Hibi, et al., Genes & Dev., 7:000, 1993).

While the intensity of spot b, a doubly phosphorylated tryptic peptidecontaining the C-terminal phosphorylation sites of c-Jun (Boyle, et al.,Cell, 64:573-584, 1991; Lin, et al., Cell, 70:777-789, 1992), was moreor less invariant, TPA treatment resulted in a small increase in theintensity of the monophosphorylated form of this peptide (spot c) at theexpense of the triple phosphorylated form (spot a). This effect was alsoobserved in response to costimulation with TPA+A23187. In contrast toHeLa cells and fibroblasts (Boyle, et al., supra, 1991; Minden, et al.,Nature, 1993), TPA treatment of Jurkat cells resulted in increasedphosphorylation of the N-terminal sites, corresponding to Ser63 (spot y)and Ser73 (spot x) and this effect was strongly enhanced by A23187. CsAprevented the enhancement of N-terminal phosphorylation by A23187.

EXAMPLE 12 Synergistic Activation of JNK

Studies were done to determine whether enhanced N-terminal c-Junphosphorylation in response to TPA+A23187 was due to synergisticactivation of JNK, the protein kinase that binds to c-Jun andphosphorylates its N-terminal sites. JNK exists in two forms, 46 kD and55 kD in size, both of which are activated by external stimuli (Hibi, etal., supra, 1993; Deng, et al., supra, 1993). In-gel kinase assaysindicated that both forms of JNK were activated by TPA (FIG. 13A). Wholecell extracts (WCE) of Jurkat cells incubated with TPA (T, 50 ng/ml),A23187 (A, 1 μg/ml) or CsA (100 ng/ml) for 15 minutes, alone or incombination, were separated by SDS-PAGE (100 μg protein/lane) on gelsthat were cast in the absence or presence of GST-cJun (1-223). The gelswere subjected to renaturation protocol and incubated in kinase buffercontaining γ-³²P-ATP. The protein bands corresponding to the 55 kD and46 kD forms of JNK are indicated.

While A23187 treatment by itself did not activate JNK, it potentiatedits activation by TPA. CsA blocked this costimulatory effect.

JNK can be retained on GSTcJun-glutathione (GSH) agarose affinity resinand its kinase activity measured by phosphorylation of GSTcJun. WCE (50μg) of Jurkat cells treated as described above were incubated with 5 μlof GSH agarose beads coated with 10 μg GST-cJun (1-223) for 12 hours at4° C. After extensive washing, the beads were incubated in kinase buffercontaining γ-³²P-ATP for 20 minutes at 30° C., after which the proteinswere dissociated by incubation in SDS sample buffer and separated bySDS-PAGE (FIG. 13B). The 49 kD band corresponds to GST-cJun (1-223). Thefaster migrating bands are degradation products (Hibi, et al., supra,1993).

This solid-state assay also indicated that TPA treatment resulted inactivation of JNK, which was strongly potentiated by A23187, which byitself had no effect. This synergistic activation of JNK was inhibitedby CsA (FIG. 13B). To prove that the solid-state assay measures theactivity of the same polypeptides identified by the in-gel kinase assay,JNK was first isolated on GSTcJun-GSH agarose beads and then analyzed itby an in-gel kinase assay. Both the 55 and 46 kD forms of JNK bound toGSTcJun and were regulated in the same manner revealed by the bindingassay (FIG. 13C) WCE (200 μg) of Jurkat cells treated as described inFIG. 13A were incubated with GST-cJun(1-223)-GSH agarose beads asdescribed above and the bound fraction was eluted in SDS sample bufferand separated by SDS-PAGE on a gel containing GST-cJun(1-223). The gelwas renatured and incubated in kinase buffer containing γ-³²P-ATP tolabel the JNK polypeptides.

EXAMPLE 13 Costimulation by Ca⁺⁺ is Unique to JNK and T Lymphocytes

We examined whether elevated intracellular Ca⁺⁺ affects JNK activationin other cells. JNK activity was weakly stimulated by TPA in CV1 andFR3T3 cells, but not in PC12 cells (FIG. 14). Cultures of FR3T3, CV-1,PC12 and mouse thymocytes were incubated for 15 minutes in the presenceof TPA (50 ng/ml, T), A23817 (1 μg/ml, A) and/or CsA (100 ng/ml), asindicated. WCE prepared from 2-4×10⁵ cells for the established celllines and 1.5×10⁶ cells for primary thymocytes were incubated withGSTcJun(1-223)-GSH agarose beads. After washing, JNK activity wasdetermined by solid-state phosphorylation assay as described above.

In none of these cells was JNK activity affected by A23187 or CsAtreatment. Similar results were obtained in HeLa, HepG2 and Gc cells. Bycontrast, the regulation of JNK activity in mouse thymocytes was similarto that observed in Jurkat cells. TPA induced a moderate increase in JNKactivity which was enhanced by A23187 and that costimulation wasinhibited by CsA (FIG. 14).

JNK is a proline-directed protein kinase activated by extracellularstimuli (Hibi, et al., supra, 1993). In that respect, it resembles theERK1 and 2 MAP kinases (Boulton, et al., Cell, 65:663-675, 1991). SinceERK1 and 2 appear to be involved in induction of c-fos (Gille, et al.,Nature, 358:414-417, 1992; Marais, et al., Cell, 73:381-393, 1993) andcould thereby participate in T cell activation, their regulation wasexamined. ERK1 and ERK2 activities were measured in both Jurkat andmouse thymocytes using an immunecomplex kinase assay and myelin basicprotein (MBP) as a substrate. Recombinant, kinase-defective ERK1 wasalso used a substrate for assaying MEK, the protein kinase responsiblefor activation of ERK1 and 2 (Crews, et al., Science, 258:478-480,1992). Both ERK and MEK activities were fully stimulated by TPAtreatment of either Jurkat cells or mouse thymocytes (FIG. 15).

WCE (5 μg) of Jurkat (FIG. 15, panel A) or mouse thymocytes (panel C)were incubated with 1 μg of kinase-defective ERK1 in kinase buffercontaining γ-³²P-ATP for 20 minutes. The phosphorylated proteins wereseparated by SDS-PAGE and the band corresponding to the mutant ERK1 isindicated. WCE (20 μg) of Jurkat (panel B) or mouse thymocytes (panel C)that were treated as described above were immunoprecipitated withanti-ERK antibodies (a gift from Dr. M. Weber). The immune complexeswere washed and incubated in kinase buffer containing γ-³²P-ATP and 2 μgMBP for 15 minutes at 30° C. The phosphorylated proteins were separatedby SDS-PAGE. The band corresponding to phosphorylated MBP is indicated.A23187 and CsA had no effect on either activity.

EXAMPLE 14 Synergistic Activation of JNK by Anti-CD3 and Anti-CD28

If JNK plays a central role in signal integration during T cellactivation, then other costimulatory paradigms should also cause itssynergistic activation. The regulation of JNK in response to T cellactivation with anti-CD3 and anti-CD28 antibodies was examined. Jurkatcells (1×10⁷) were incubated for 15 minutes with either normal mouseserum, 1 μg/ml anti-CD3 and/or 2 μg/ml anti-CD28, in the absence orpresence of 100 ng/ml CsA, as indicated. WCE were prepared and 100 μgsamples were analyzed for JNK activation using the in-gel kinase assay,as described above.

While incubation of Jurkat cells with either soluble anti-CD3 or solubleanti-CD28 alone had a negligible effect on JNK activity, simultaneousincubation with both antibodies resulted in strong synergisticactivation of both forms (FIG. 16A).

WCE (50 μg) of Jurkat cells treated as described above were incubatedwith GSTcJun(1-223)-GSH agarose beads and assayed for JNK activity usingthe solid-state kinase assay. The same WCE (20 μg) wereimmunoprecipitated with anti-ERK2 antibodies and assayed for MBP-kinaseactivity. CsA partially attenuated this effect. By contrast, incubationwith soluble anti-CD3 was sufficient for efficient activation of ERK2,which was not enhanced by costimulation with anti-CD28, nor was itinhibited by CsA (FIG. 16B).

To further investigate the nature of signal integration by JNK, theeffect of a suboptimal dose of TPA was examined, which by itself doesnot lead to JNK activation on the responses to either anti-CD3 oranti-CD28 (FIG. 16C). WCE (50 μg) of Jurkat cells treated as describedin Panel A with various stimuli alone or their combinations wereincubated with GSTcJun(1-223)-GSH agarose beads and assayed for JNKactivity using solid-state kinase assay. The same samples (20 μg) werealso assayed for MBP-kinase activity as described in FIG. 16B.

Together with A23187, this suboptimal dose of TPA resulted in a strongsynergistic activation of JNK but not ERK2. The activation of JNK wascompletely inhibited by CsA. The suboptimal dose of TPA also led tostrong synergistic activation of JNK together with either anti-CD3 oranti-CD28. ERK2, on the other hand, was fully activated by anti-CD3 andsuboptimal TPA, which by itself led to partial activation of ERK2, hadno further effect. Exposure to anti-CD28 did not augment the activationof ERK2 by TPA. JNK was also efficiently activated by combined treatmentwith anti-CD3 +A23187, but not by anti-CD28+A23187.

EXAMPLE 15 Activation of Ha-Ras

The effects of the various treatments on Ha-Ras activation were examinedand the results shown in FIG. 17. Jurkat cells (2×10⁶ cells per point)labeled with 0.4 mCi of ³²P-orthophosphate for 3 hours were incubatedwith nonspecific antibody (1 μg/ml mouse IgG; control), 1 μg/mlanti-CD3, 2 μg/ml anti-CD28, 10 ng/ml TPA or 500 ng/ml A23187 (A), asindicated. After 2 minutes, the cells were harvested, lysed and Ha-Raswas isolated by immunoprecipitation. The guanine nucleotide bound toHa-Ras were extracted, separated by thin layer chromatography andquantitiated as described in EXAMPLE 9. The values shown represent theaverages of two separate experiments done in duplicates. Jurkat cellswere labeled with ³²P-orthophosphate and stimulated with either TPA oranti-CD3 as described above. At the indicated time points, the cellswere harvested and the GTP content of Ha-Ras was determined as describeddirectly above.

Whereas an optimal dose of TPA and exposure to soluble anti-CD3 led toactivation of Ha-Ras, measured by an increase in its GTP content,soluble anti-CD28 had no effect on Ha-Ras activity (FIG. 17A). Theactivation of Ha-Ras by either anti-CD3 or TPA was not augmented bycostimulation with either anti-CD28 or A23187, respectively. While theactivation of Ha-Ras by TPA persisted for at least 20 minutes, theresponse to soluble anti-CD3 was highly transient (FIG. 17B). Therefore,signal integration must occur downstream of Ha-Ras.

EXAMPLE 16 Cloning of JNK1 Polynucleotide (46 kD)

To identify novel members of the MAP kinase group, a polymerase chainreaction (PCR) strategy was employed using degenerate primers to amplifysequences from a human liver cDNA library.

cDNA Cloning

Degenerate oligonucleotides CAYMGNGAYNTNAARCC (SEQ ID NO: 13) andGAGAGCCCATNSWCCADATR TC (SEQ ID NO: 14) were designed based on conservedkinase sub-domains and employed as PCR primers to isolate fragments ofMAP kinase-related cDNAs from a human liver cDNA library. Comparison ofthe sequence of 387 clones with the GenBank database (Blast Fileserver,National Center for Biotechnology Information) allowed identification ofone clone that exhibited a high level of homology with members of theMAP kinase family. This partial cDNA was used to screen a λZapI humanfetal brain cDNA library (Stratagene Inc., La Jolla, Calif.). Threepositive clones were obtained after screening 10⁶ phage. DNA sequencingof both strands of each clone was performed using a PCR procedureemploying fluorescent dideoxynucleotides and a model 373A automatedsequencer (Applied Biosystems). This analysis demonstrated that theseclones corresponded to overlapping cDNAs. The sequence of the largestclone (1418 bp) includes the complete JNK1 coding region and is shown inFIGS. 18A and D. A single long open reading frame that encodes aputative protein kinase, JNK1, with a predicted mass of 44.2-kDa wasidentified. In-frame stop codons in the 5′ and 3′ regions of the cDNAindicate that this clone contains the entire JNK1 coding region. FIG.18B shows a comparison of the deduced sequence of JNK1 with other MAPkinases.

The deduced sequence of JNK1 is aligned with those of the MAP kinasesHOG1 (Brewster, et al., Science 259:1760-1763, 1993), MPK1 (Torres, etal., Mol. Microbiol. 5:2845-2854, 1991; Lee, et al., C. Mol. Cell. Biol.13:3067-3075, 1993), FUS3 (Elion, et al., Cell 60:649-664, 1990), KSS1(Courchesne, et al., Cell 58:1107-1119, 1989), ERK1 (Boulton, et al.,Science 249:64-67, 1990) and ERK2 (Boulton, et al., Cell 65:663-675,1991) using the PILEUP program (Wisconsin Genetics Computer Group). Gapsin the sequences that were introdued to optimize the alignment areillustrated with a dash (−). Residues that are identical are indicatedwith a period (.). The carboxyl termini of HOG1 and MPK1 that extendbeyond the kinase domain are truncated (>). The protein kinasesub-domains located within the deduced protein sequence are illustratedand the conserved tyrosine and threonine phosphorylation sites (*) areindicated with asterisks (Davis, J. Biol. Chem 268:14553-14556, 1993).

Comparison of the deduced structure of JNK1 with the Genbank data-base(Blast Fileserver, National Center for Biotechnology Information)revealed homology to the MAP kinases ERK1 (Boulton, et al., Science249:64-67, 1990) and ERK2 (Boulton, et al., Cell 65:663-675, 1991).Sequence homology was also observed between JNK and the yeast MAPkinases HOG1 (Brewster, et al., Science 259:1760-1763,1993), MPK1(Torres, et al., Mol. Microbiol. 5:2845-2854, 1991; Lee, et al., C. Mol.Cell. Biol. 13:3067-3075, 1993), FUS3 (Elion, et al., Cell 60:649-664,1990), and KSS1 (Courchesne, et al, Cell 58:1107-1119, 1989).Significant regions of identity between JNK1 and other MAP kinases arefound throughout the protein kinase domain. Notably, the Thr and Tyrphosphorylation sites located in sub-domain VIII, that are required forMAP kinase activation (Payne, et al., EMBO J. 10:885-892, 1991), areconserved in JNK1. Together, these sequence similarities indicate thatJNK1 is a distant relative of the MAP kinase group (FIG. 18C).

FIG. 18C shows the comparison which was created by the PILEUP programusing a progressive pair-wise alignment and shown as a dendrogram. Theidentity of the kinases with JNK1 was calculated with the BESTFITprogram: ERK1 (39.7%); ERK2 (43.1%); HOG1 (41.1%); FUS3 (41.5%); KSS1(40.6%); MPK1 (41.0%); SPK1 (40.1%); CDC2 (37.5%); GSK-3a (29.7%);protein kinase Aa (21.5%); and protein kinase Ca (22.6%). The similarityof the kinases to JNK was calculated with the BESTFIT program: ERK1(64.5%); ERK2 (67.6%); HOG1 (64.2%); FUS3 (63.9%); KSS1 (63.9%); MPK1(63.7%); SPK1 (63.5%); CDC2 (58.7%); GSK-3a (50.6%); protein kinase Aa(48.8%); and protein kinase Ca (44.2%). The PILEUP and BESTFIT programswere from the Wisconsin Genetics Computer Group.

EXAMPLE 17 Localization of JNK mRNA

To examine the tissue distribution of JNK1, Northern blot analysis wasused.

Hybridization Analysis

Northern blots were performed using 2 μg of polyA⁺ RNA isolated fromdifferent human tissues, fractionated by denaturing agarose gelelectrophoresis and transferred onto a nylon membrane (Clontech). Theblots were hybridized to a probe that was prepared by labeling the JNK1cDNA with [α-³²P]dCTP (Amersham International PLC) by random priming(Stratagene Inc.). The integrity of the mRNA samples was confirmed byhybridization to an actin probe. Southern blot analysis was performedusing 10 μg of human genomic DNA that was digested with differentrestriction enzymes, fractionated by agarose gel electrophoresis andtransferred onto a nylon membrane. The membrane was probed with arandom-primed fragment of JNK1 cDNA (797 bp to 1275 bp). The blots werewashed three times with 1×SSC, 0.05% SDS, and 1 mM EDTA prior toautoradiography.

A single major JNK1 transcript (3.5 Kb) was observed in fetal brain(FIG. 19A). In adult tissues there was ubiquitous expression oftranscripts that hybridized to the JNK1 probe. However, atissue-specific heterogeneity of the mRNA was observed in adult tissues(FIG. 19B). This heterogeneity could result from alternative processingof transcripts from a single gene. Alternatively, it is possible thatJNK1 represents the prototype for a sub-family of closely-relatedprotein kinases. Consistent with this hypothesis is the observation ofmultiple bands that hybridized to a JNK1 probe during Southern blotanalysis of human genomic DNA (FIG. 19C). Human genomic DNA digestedwith different restriction enzymes was examined by Southern blotanalysis using a JNK1 cDNA probe. The genomic DNA was restricted withEcoRI (lane 1), HindIII (lane 2), BamHI (lane 3), PstI (lane 4), andBgIII (lane 5). The position of DNA size markers in kilobases isillustrated.

EXAMPLE 18 JNK1 is Activated During the UV Response

To characterize the kinase activity of purified JNK1, an expressionvector encoding an epitope-tagged JNK1 protein that could beimmunoprecipitated using a monoclonal antibody was constructed.

The JNK1 cDNA was first cloned into the expression vector pCMV5(Andersson, et al., J. Biol. Chem. 264:8222-8229, 1989) between the XbaIand HindIII sites. A PCR-based procedure was employed to insert anepitope tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (SEQ ID NO: 15) betweencodons 1 and 2 of the JNK1 cDNA (Ho, et al., Gene 77:51-59, 1989). Asimilar method was employed to insert an HA epitope-tag. Substitution ofthe phosphorylation sites Thr-183 and Tyr-185 by Ala and Phe,respectively, was performed by cassette mutagenesis using a degeneratedouble-stranded oligonucleotide and the PstI and StyI restriction sites.The sequence of these constructs was confirmed by automated sequencingwith a model 373A DNA sequencer (Applied Biosystems).

The plasmid pCMV-Ras/Leu61 was provided by Dr. L. Kozma (University ofMassachusetts Medical School). The plasmids encoding GST-Jun fusionproteins were described previously (Hibi, et al., Genes Dev.7:2135-2148, 1993). Plasmid DNA (1 μg) was transfected into COS-1 cellsusing the lipofectamine method (Gibco-BRL). After 48 hours, the cellswere treated without and with TPA, EGF or UV-C.

JNK1 protein kinase activity was detected in the immune-complex.Immunecomplex kinase assays using either M2 immunoprecipitates orpurified JNK1, JNK-46, and ERK1 or ERK2 were performed at 30° C. for 20mins using 3 μg of substrate, 20 μM ATP and 5 μCi of [γ-³²P]ATP in 30 μlof kinase buffer (25 mM Hepes (pH 7.6), 20 mM MgCl₂, 20 mMβ-glycerophosphate, 20 mM p-nitrophenyl phosphate, 0.1 mM Naorthovanadate, 2 mM DTT). The reactions were terminated with Laemmlisample buffer and the products were resolved by SDS-PAGE (12% gel). JNK1protein activity was also measured after SDS-PAGE by the in-gel kinaseassay with the substrate GST-cJun(1-79) as described by Hibi, et al.,supra, (1993). Solid-phase protein kinase assays were performed asdescribed by Hibi, et al., supra, (1993). Clarified cell extracts wereincubated with GST fusion proteins immobilized on GSH-agarose beads.After 3 hours at 4° C., the beads were washed extensively and bound JNK1was detected by the addition of [γ-³²P]ATP. The reaction was terminatedafter 10 mins at 30° C. and the products were resolved by SDS-PAGE. Theincorporation of [³²P]phosphate was visualized by autoradiography andquantitated with a phosphorimager and ImageQuant software (MolecularDynamics Inc., Sunnyvale, Calif.). The methods used for phosphopeptidemapping (Boyle, et al., Cell 64:573-584, 1991) and phosphoamino acidanalysis (Alvarez, et al., J. Biol. Chem 266:15227-15285, 1991) weredescribed previously.

In initial studies designed to characterize JNK1 activity, SDS-PAGE andan in-gel kinase assay were used to identify the apparent mass of theJNK1 protein kinase. Essentially identical results were obtained usingstandard immune-complex kinase assays. Autophosphorylation of JNK1 wasnot observed in experiments performed in the absence of an exogenoussubstrate. However, a low level of kinase activity migrating at 46-kDawas detected when a recombinant fragment of the c-Jun activation domain(GST-cJun(1-79)) was used as a substrate (FIG. 20A).

Epitope-tagged JNK1 was expressed in COS cells. Control experiments wereperformed using mock-transfected cells. After 48 hours, the cells weretreated either without or with 100 nM TPA, 10 nM EGF, or 80 J/m² UV-Cand incubated for 1 hr. The cells were lysed in RIPA buffer and the JNK1proteins were isolated by immunoprecipitation with the M2 monoclonalantibody. JNK1 protein kinase activity was measured after SDS-PAGE usingan in-gel kinase assay with the substrate GST-cJun(1-79) polymerizedinto the gel.

COS-1 cells were maintained in. Dulbecco's modified Eagle's mediumsupplemented with 5% bovine serum albumin (Gibco-BRL). Metaboliclabeling with [³²P]phosphate was performed by incubation of cells inphosphate-free modified Eagle's medium (Flow Laboratories Inc.)supplemented with 0.1% fetal bovine serum and 1 mCi/ml[³²P]orthophosphate (Dupont-NEN). COS cells were treated with 10 nM EGFor 100 nM thorbol myristate acetate. The cells were then incubated fordefined times at 37° C. prior to harvesting and measurement of JNK1protein kinase activity. The data are presented as arbitrary units.Treatment of the transfected cells with EGF or phorbol ester (TPA)caused a low level of JNK1 activation that was sustained forapproximately 2 hours (FIG. 20B). In contrast, exposure to UV radiationcaused a marked increase in JNK1 activity. Significantly, theelectrophoretic mobility of the UV-stimulated JNK1 enzymatic activity issimilar to JNK-46 (Hibi, et al., supra, 1993). The slightly slowermobility of JNK1 compared with JNK-46 is most likely caused by theoctapeptide epitope-tag fused to JNK1.

The UV-dose response was examined by exposing COS cells to UV-Cradiation and the cells were harvested after incubation for 1 hr. Thetime course was investigated by exposure of COS cells to 40 J/m² UV-Cand then incubating the cells for defined times. JNK1 activity wasmeasured by immunoprecipitation with the M2 monoclonal antibody, in-gelkinase assay, and phosphorimager detection.

Phosphorimager Detection

Metabollically labeled cells were lysed in 25 mM Hepes (pH 7.5), 1%Triton X-100, 1% (w/v) deoxycholate, 0.1% (w/v) SDS, 0.5 M NaCl, 50 mMNaF, 1 mM Na orthovanadate, 5 mM EDTA, 10 μg/ml leupeptin, 1 mM PMSF.Soluble extracts were prepared by centrifugation at 100,000×g for 30mins at 4° C. The extracts were pre-cleared using protein G-Sepharose(Pharmacia-LKB Biotechniologies Inc.) and then incubated with themonoclonal antibody M2 (IBI-Kodak) pre-bound to protein G-Sepharose. TheM2 antibody recognizes the epitope Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(Flag; Immunex Corp.) and immunoprecipitates the epitope-tagged JNK1protein. (In some experiments the monoclonal antibody 12CA5 was used toimmunoprecipitate JNK1 tagged with the HA epitope). After 1 hr ofincubation, the immunoprecipitates were washed three times with lysisbuffer and once with 25 mM Hepes (pH 7.5), 0.2% (w/v) Triton X-100, 1 mMEDTA.

Prior to analysis by Western blotting, protein samples were resolved bySDS-PAGE and electroblotted onto immobilon P membranes (Millipore). Themembranes were probed with the monoclonal antibody M2 (IBI-Kodak) andimmunecomplexes were visualized using enhanced chemiluminescencedetection (Amersham International PLC).

The UV-induced activation of JNK1 occured rapidly after UV-irradiationwith maximal activation at 1 hr followed by a progressive decline inJNK1 activity at later times (FIG. 20C). Examination of the UVdose-response revealed detectable JNK1 activation at 20 J/m² and maximalactivation at approximately 80 J/m² (FIG. 20D). Significantly, thetime-course and dose-response of JNK1 activation by UV (FIG. 20) wassimilar to the regulation of the endogenous JNK-46 protein kinaseexpressed by COS cells (FIG. 21). FIG. 21 shows the time course and doseresponse of UV activation of endogenous JNK1 expressed by COS cells. TheUV dose-response was examined by exposing COS cells to UV-C radiationand the cells were harvested after 1 hr. The time-course wasinvestigated by exposure of COS cells to 40 J/m² UV-C and thenincubating the cells for defined times. Endogenous JNK1 activity wasmeasured using the solid-phase kinase assay with the substrateGST-cJun(1-79) as described.

The electrophoretic mobility of JNK1, its potent activation by UV, thelack of detectable autophosphorylation, and the efficientphosphorylation of GST-cJun fusion proteins suggests that JNK1 ishomologous or identical to the protein kinase activity JNK-46 that hasbeen identified in UV-irradiated cells (Hibi, et al., supra, 1993).

EXAMPLE 19 Ha-Ras Activates JNK1 and Potentiates the UV Response Pathway

The results of previous studies have shown that oncogenically activatedRas stimulates the NH₂-terminal phosphorylation of c-Jun (Binetruy, etal., supra., 1991; Smeal, et al., supra., 1991; 1992). In addition, Rasis involved in the UV-response leading to increased c-Jun activity(Devary, et al., supra, 1992; Radler-Pohl, et al., EMBO J. 12:1005-1012,1993). The effect of oncogenically activated Ras on JNK1 was studied.Epitope-tagged JNK1 was coexpressed in COS cells without or withactivated Ha-Ras. After 48 hours, the cells were exposed to differentdoses of UV-C and then incubated for 1 hr at 37° C. JNK1 was isolated byimmunoprecipitation with the M2 monoclonal antibody and JNK1 activitywas measured using an immunecomplex kinase assay with the substrateGST-cJun(1-79).

Significantly, expression of activated Ha-Ras potentiated UV-stimulatedJNK1 activity (FIG. 22). By itself, Ha-Ras caused JNK1 activation thatwas approximately 40% of that obtained with 40 J/m² UV-irradiation.These data indicate that Ha-Ras partially activates JNK1 and that Ha-Raspotentiates the activation caused by UV. JNK1 was expressed in COS cellsand activated by exposure to UV light. JNK1 was isolated byimmunoprecipitation with the M2 monoclonal antibody and used tophosphorylate 3 μg of GST (control, lane 1), GST-cJun(1-223) (lane 2),GST-cJun(43-233) (lane 3), GST-cJun(1-79) (lane 4),GST-cJun(1-223/Ala63, Ala-73) (lane 5), GST-chcJun(1-144) (chickenc-Jun, lane 6), GST-chvJun(1-144) (chicken v-Jun, lane 7), or MBP (lane8). After the phosphorylation reaction, the different proteins wereseparated by SDS-PAGE and visualized by autoradiography. The sameproteins were used as substrates for JNK-46 purified from UV-irradiatedHeLa cells (B) or a mixture of purified ERK1 and ERK2 (C). TheCoomassie-blue stain of the protein substrates is also shown (D). Themigration positions of the full-length substrate proteins are indicatedby the dots.

EXAMPLE 20 JNK1 Phosphorylates C-JUN at SER-63 and SER-73

To investigate the relationship between JNK1 and JNK-46, the substratespecificity of JNK was examined.

FIG. 23 (panel A) shows that both GST-vJun and myelin basic protein(MBP) are very poor substrates for JNK1, while GST-cJun fusion proteinsare excellent JNK1 substrates. This pattern of substrate specificity isidentical to JNK-46 purified from UV-irradiated HeLa cells (FIG. 23,panel B). Like JNK-46, JNK1 efficiently phosphorylated GST-cJun fusionproteins containing residues 1-223 and 1-79 of c-Jun. However, adeletion of c-Jun NH₂-terminal sequences including the d-domain(residues 1-42) caused a marked decrease in phosphorylation by JNK1.Replacement of Ser-63 and Ser-73 with Ala also decreased the observedphosphorylation. On the other hand, the substrate specificity of the MAPkinases ERK1 and ERK2 was markedly different from that of JNK1 (FIG. 23,panel C). In this-case myelin basic protein (MBP) was a significantlybetter substrate than GST-cJun. In addition, there was no discriminationbetween GST-cJun, GST-vJun, and the mutants lacking JNK1 phosphorylationsites [GST-cJun(Ala)] or the JNK1 binding site [GST-CJun(43-223)]. TheCoomasie blue stain of the protein substrates is also shown (FIG. 23,panel D).

To further establish the substrate specificity of JNK1, the sites ofc-Jun phosphorylation were determined by phosphopeptide mapping.GST-cjun(1-223), GST-cJun(1-223/Ala63,73), GST-cJun(1-79) andfull-length c-Jun were phosphorylated by epitope-tagged JNK1immunopurified from UV-irradiated transfected COS cells. Full-lengthc-Jun was also phosphorylated by JNK-46 purified from UV-irradiated HeLacells. The phosphorylated proteins were isolated by SDS-PAGE, elutedfrom the gel, and digested with trypsin. The tryptic digests wereseparated by thin layer electrophoresis (horizontal dimension) followedby ascending chromatography (vertical dimension) and visualized byautoradiography. The origin and the phosphopeptides X, Y, T1, and T2 areindicated.

The major phosphopeptides observed were X and Y (FIG. 24). Thesephosphopeptides were also found in maps of c-Jun phosphorylated bypurified JNK-46. In previous studies, these phosphopeptides were shownto correspond to the phosphorylation of the regulatory sites Ser-63 andSer-73 (Binetruy, et al., Nature 351:122-127, 1991; Pulverer, et al.,Nature 353:670-674, 1991; Smeal, et al., supra, 1991; 1992). Examinationof the primary sequence surrounding these phosphorylation sitesindicates the consensus sequence motif Leu/Ala-Ser*-Pro-Asp/Glu (SEQ IDNO: 16). A low level phosphorylation of sites other than Ser-63 andSer-73 was also observed, and was not affected by substitution of Ser-63and Ser-73 with Ala (phosphopeptides T1 and T2). These sites correspondto phosphorylation at Thr-Pro motifs (Thr-91, Thr-93, or Thr-95) thatwere previously identified as minor JNK1 sites (Hibi, et al., supra,1993). Importantly, no phosphorylation of the COOH-terminal sites(Boyle, et al., Cell 64:573-584, 1991; Lin, et al., Cell70:777-789,1992) including Ser-243, which is phosphorylated by purifiedERKs (Alvarez et al., J. Biol. Chem. 266:15227-15285, 1991) wasobserved. The low level of COOH-terminal phosphorylation previouslyobserved with partially purified JNK1 preparations (Hibi, et al., supra,1993) is most likely due to contamination with other protein kinases.

EXAMPLE 21 JNK1 Associates With the c-JUN Transactivation Domain

The observation that v-Jun is not a good JNK1 substrate is intriguingbecause both v-Jun and c-Jun contain the phosphoacceptor sites Ser-63and Ser-73. This suggests that the presence of the primary sequenceencoding a phosphorylation site may be insufficient for efficientsubstrate recognition by JNK1. In previous studies a small region of thec-Jun transactivation domain, the δ sub-domain (Vogt, et al., Adv.Cancer Res. 55:1-35, 1990), was proposed to mediate the directinteraction of c-Jun with a physiologically relevant protein kinase thatphosphorylates Ser-63 and Ser-73 (Adler, et al., Proc. Natl. Acad. Sci.USA, 89:5341-5345, 1992) and was identified as a binding site for JNK1.According to this hypothesis, the inefficient phosphorylation of v-Junis due to defective JNK1 binding.

To investigate whether JNK1 is a physiologically relevant c-Jun proteinkinase, the binding of immunopurified JNK1 isolated from UV-stimulatedCOS cells was studied. The binding of JNK1 to c-Jun was detected using asolid-phase kinase assay in which JNK1 binds to an immobilized GST-cJunfusion protein and, after the addition of ATP, phosphorylates Ser-63 andSer-73.

COS cells expressing epitope-tagged JNK1 were lysed in Buffer A and asoluble extract was obtained after centrifugation at 100,000×g for 20mins. COS cell extracts (250 μg) were incubated with 20 μg GST orGST-cJun immobilized on 10 μl GSH-agarose at 4° C. for 5 hours. Thebeads were washed four times with Buffer A and JNK1 was eluted withLaemmli sample buffer.

Detection of JNK1 binding to the c-Jun transactivation domain wasexamined using a solid-state protein kinase assay. Epitope-tagged JNK1was expressed in COS cells and activated by UV-irradiation.Mock-transfected COS cells were used as a control. After 1 hour, thecells were lysed and subjected to immunoprecipitation with the M2monoclonal antibody. The immunecomplexes were eluted with urea and,after dialysis, the isolated JNK1 was incubated with GST-Jun fusionproteins bound to GSH-agarose beads. The beads were washed extensivelyand bound JNK1 was detected using a solid-phase kinase assay asdescribed. The phosphorylated proteins were resolved by SDS-PAGE anddetected by autoradiography. The dots indicate the migration of GST andthe different GST-cJun proteins (see FIG. 25A).

To examine whether the binding of JNK1 to c-Jun was altered by the stateof JNK1 activation, the binding of epitope-tagged JNK1 to immobilizedc-Jun was examined by Western blotting. JNK1 from either unstimulated orUV-irradiated cells bound to GST-cJun. Epitope-tagged JNK1 was expressedin COS cells (lanes 3-5). Mock-transfected COS cells were used incontrol experiments (lanes 1 & 2). The cells were either untreated(lanes 1 & 3) or irradiated with 40 J/m² UV-C (lanes 2, 4, & 5). Celllysates were prepared and incubated with GST-cJun(1-79) (lanes 1-4) orGST (lane 5) immobilized on GSH-agarose beads. The beads were washedextensively and bound JNK1 was detected by Western blot analysis usingthe M2 monoclonal antibody. Lane 6 represents an unfractionated lysateof cells expressing JNK1 (see FIG. 25B).

Deletion of residues 1-42 or 1-55 of the c-Jun transactivation domain,which includes the γ sub-domain, prevented binding of JNK1 (FIG. 25A).Deletion of c-Jun residues 1-32 reduced, but did not eliminate, JNK1binding. However, deletion of residues 1-22 had no effect on JNK1binding. Previous studies have demonstrated that the effect of thesedeletions on GST-cJun phosphorylation is due to changes in JNK1 bindingrather than the presentation of the phosphoacceptor sites. Takentogether, these observations demonstrate that a region of the c-JunNH₂-terminus adjacent to the major phosphorylation sites (Ser-63 andSer-73) is required for binding to JNK1.

These data demonstrate that JNK1 binds to a specific region (residues23-79) of the NH₂-terminal transactivation domain of c-Jun. It is likelythat this binding reflects the direct interaction of JNK1 with c-Jun.However, these experiments do not exclude the possibility that anaccessory protein is required for JNK1 binding to c-Jun or that anaccessory protein may stabilize this interaction.

EXAMPLE 22 Phosphorylation at Thr and Tyr is Required for UV-InducedJNK1 Activation

MAP kinases are activated by dual phosphorylation at Thr and Tyrresidues within sub-domain VIII. Therefore tested whetherphosphorylation at these sites is required for JNK1 activation by UV.The predicted phosphorylation sites Thr-183 and Tyr-185 were replaced byAla and Phe, respectively, using site-directed mutagenesis.

FIG. 26, panels A, B and C show the results indicating that substitutionof Thr-183 or Tyr-185 blocks the UV-stimulated phosphorylation of JNK1.Site-directed mutagenesis was used to replace the JNK1 phosphorylationsites Thr-183 with Ala and Tyr-185 with Phe. The epitope-taggedwild-type JNK1 (TPY) and the mutated JNK1 proteins (APY, TPF, and APF)were expressed in COS cells. The cells were either exposed or notexposed to 80 J/m² UV-C, incubated for 1 hr at 37° C., and then lysed inRIPA buffer. JNK1 was isolated by immunoprecipitation with the M2monoclonal antibody and SDS-PAGE. The level of expression of thewild-type and mutated JNK1 proteins was examined by Western blotanalysis using the M2 monoclonal antibody and enhanced chemiluminescencedetection (FIG. 26A). The phosphorylation state of JNK1 was examinedusing cells metabolically-labeled with [³²P]phosphate (FIG. 26B). Thephosphorylated JNK1 proteins were subjected to phosphoamino acidanalysis (FIG. 26C).

JNK1 protein kinase activation by UV is inhibited by substitution ofTir-183 or Tyr-185 as shown in FIG. 26D. Epitope-tagged JNK proteinswere immunoprecipitated from COS cell lysates using the M2 monoclonalantibody. JNK1 protein kinase activity was measured after SDS-PAGE usingan in-gel kinase assay with the substrate GST-cJun(1-79).

A similar level of expression of the wild-type and mutated JNK1 proteinswas obtained in transiently transfected COS cells (FIG. 26A). JNK1phosphorylation was examined using cells metabolically-labeled with[³²F]phosphate. FIG. 26B shows that UV-treatment caused increasedphosphorylation of wild-type JNK1. Phosphoamino acid analysisdemonstrated a high level of basal serine phosphorylation of JNK1 (FIG.26C). The UV-stimulated phosphorylation of JNK1 was accounted for byincreased [³²P]phosphothreonine and [³²P]phosphotyrosine (FIG. 26C).

F9 cells were transfected with 10 μg of expression vector encodingepitope-tagged (HA) JNK1. The cells were labeled 12 hours aftertransfection for 4 hours with [³²P]phosphate (0.5 mCi/ml). One dish wasexposed to 100 J/m² UV-C and the cells were harvested 45 mins later.HA-JNK1 was purified by immunoprecipitation (12CA5 antibody) andSDS-PAGE. Phosphorylated JNK1 was eluted from the gel and digested withtrypsin. The tryptic digests were separated by thin layerelectrophoresis (horizontal dimension) followed by ascendingchromatography (vertical dimension) and visualized by autoradiography (9days at −80° C.). The constitutive phosphopeptide (C), the inducedphosphopeptide (I), and the origin (arrow head) are indicated in FIG.27.

Tryptic phosphopeptide mapping demonstrated that this dualphosphorylation was associated with the appearance of one major [³²P]phosphopeptide (FIG. 27). The presence of this novel phosphopeptide isconsistent with the identification of Thr-183 and Tyr-185 as the sitesof UV-stimulated phosphorylation of JNK1. This hypothesis was confirmedby demonstrating that mutations at Thr-183 and Tyr-185 blocked theUV-stimulated phosphorylation of JNK1 (FIG. 26C). Significantly, thesemutated JNK1 proteins did not exhibit kinase activity when isolated fromeither unstimulated or UV-irradiated cells (FIG. 26D). Together, thesedata demonstrate that the mechanism of JNK1 activation involvesincreased phosphorylation at Thr-183 and Tyr-185.

EXAMPLE 23 Cloning of JNK2(55 kD)

The JNK isoform, JNK2, was molecularly cloned by screening a human HeLacell cDNA library by hybridization with a random-primed probe preparedfrom the JNK1 cDNA. The sequence of the JNK2 cDNA shown in FIG. 28,indicates that it encodes an approximately 55-kDa protein (FIG. 29) thatis related to JNK1. The cDNA is 1782 base pairs long and contains anopen reading frame from nucleotides 59 to 1330, encoding a 424 aminoacid protein (SEQ ID NO: 17 and 18, respectively). There is a high levelof protein sequence identity between JNK1 and JNK2 indicating that theseenzymes are closely related. The major sequence difference between JNK1and JNK2 is that JNK2 contains a COOH terminal extension compared withJNK1. The functional properties of JNK2 are similar to JNK1 indicatingthat these protein kinases form a group with related biologicalfunctions.

JNK2 activity was induced by UV treatment as determined by the methodsutilized for examining UV activation of JNK1, in Examples 3 and 18above. Although similarly regulated, the 46 kD polypeptide of JNK1exhibits a higher affinity for binding to c-Jun than the 55 kDpolypeptide (Example 6 and Hibi, et al., supra, 1993). The activity ofboth forms of JNK (46 and 55) is rapidly and potently stimulated by UVradiation. Although the molecular mechanisms mediating thetumor-promoting activity of UV are not completely understood, it isapparent that JNK1 and JNK2 are involved in the potentiation of AP-1activity and activated by Ha-Ras and are likely involved as mediators ofUV-induced tumor promotion.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

SEQUENCE ID LISTING

SEQ ID NO: 1 is the amino acid sequence of residues 33-79 of c-Jun.

SEQ ID NO: 2 is the nucleotide sequence for an N-terminal primer usedfor producing c-Jun truncation mutants.

SEQ ID NO: 3 is the nucleotide sequence for an N-terminal primer usedfor producing c-Jun truncation mutants.

SEQ ID NO: 4 is the nucleotide sequence for an N-terminal primer usedfor producing c-Jun truncation mutants.

SEQ ID NO: 5 is the nucleotide sequence for an N-terminal primer usedfor producing c-Jun truncation mutants.

SEQ ID NO: 6 is the nucleotide sequence for an N-terminal primer usedfor producing c-Jun truncation mutants.

SEQ ID NO: 7 is the nucleotide sequence for a C-terminal primer used forproducing c-Jun truncation mutants.

SEQ ID NO: 8 is the nucleotide sequence for a C-terminal primer used forproducing c-Jun truncation mutants.

SEQ ID NO: 9 is the nucleotide sequence and deduced amino acid sequencefor c-jun and c-Jun.

SEQ ID NO: 10 is the deduced amino acid sequence of c-Jun.

SEQ ID NO: 11 is the nucleotide sequence and deduced amino acid sequenceof JNK1.

SEQ ID NO: 12 is the deduced amino acid sequence of JNK1.

SEQ ID NO: 13 and 14 are the nucleotide sequences of degenerateoligonucleotides for cloning JNK1.

SEQ ID NO: 15 is the amino acid sequence of an epitope tag betweencodons 1 and 2 of JNK1 cDNA.

SEQ ID NO: 16 is the amino acid sequence of a consensus sequence motifsurrounding phosphorylation sites Ser-63 and Ser-73.

SEQ ID NO:17 is the nucleotide and deduced amino acid sequence of JNK2.

SEQ ID NO:18 is the deduced amino acid sequence of JNK2.

SEQ ID NO:19 is the deduced amino acid sequence of HOG1.

SEQ ID NO:20 is the deduced amino acid sequence of MPK1.

SEQ ID NO:21 is the deduced amino acid sequence of FUS3.

SEQ ID NO:22 is the deduced amino acid sequence of KSS1.

SEQ ID NO:23 is the deduced amino acid sequence of ERK1.

SEQ ID NO:24 is the deduced amino acid sequence of ERK2.

SEQ ID NO:25 is the amino acid sequence of a MAP kinase consensussequence.

25 1 47 PRT Artificial sequence Synthetic peptide 1 Ile Leu Lys Gln SerMet Thr Leu Asn Leu Ala Asp Pro Val Gly Ser 1 5 10 15 Leu Lys Pro HisLeu Arg Ala Lys Asn Ser Asp Leu Leu Thr Ser Pro 20 25 30 Asp Val Gly LeuLeu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu 35 40 45 2 35 DNA Artificialsequence PCR primer 2 tctgcaggat ccccatgact gcaaagatgg aaacg 35 3 34 DNAArtificial sequence PCR primer 3 tctgcaggat ccccgacgat gccctcaacg cctc34 4 35 DNA Artificial sequence PCR primer 4 tctgcaggat ccccgagagcggaccttatg gctac 35 5 35 DNA Artificial sequence PCR primer 5 tctgcaggatccccgccgac ccagtgggga gcctg 35 6 35 DNA Artificial sequence PCR primer 6tctgcaggat ccccaagaac tcggacctcc tcacc 35 7 30 DNA Artificial sequencePCR primer 7 tgaattctgc aggcgctcca gctcgggcga 30 8 33 DNA Artificialsequence PCR primer 8 tgaattcctg caggtcggcg tggtggtgat gtg 33 9 2096 DNAHomo Sapiens CDS (412)..(1404) 9 gaattccggg gcggccaaga cccgccgccggccggccact gcagggtccg cactgatccg 60 ctccggcgga gagccgctgc tctgggaagtcagttcgcct gcggactccg aggaaccgct 120 gcgcacgaag agccgtcagt gagtgaccgcgacttttcaa agccgggtag ggcgcgcgag 180 tcgacaagta agagtgcggg aggcatcttaattaaccctg cgctccctgg agcagctggt 240 gaggagggcg cacggggacg acagccagcgggtgcgtgcg ctcttagaga aactttccct 300 gtcaaaggct ccggggggcg cgggtgtcccccgcttgcca cagccctgtt gcggccccga 360 aacttgtgcg cgcacgccaa actaacctcacgtgaagtga cggactgttc t atg act 417 Met Thr 1 gca aag atg gaa acg accttc tat gac gat gcc ctc aac gcc tcg ttc 465 Ala Lys Met Glu Thr Thr PheTyr Asp Asp Ala Leu Asn Ala Ser Phe 5 10 15 ctc ccg tcc gag agc gga ccttat ggc tac agt aac ccc aag atc ctg 513 Leu Pro Ser Glu Ser Gly Pro TyrGly Tyr Ser Asn Pro Lys Ile Leu 20 25 30 aaa cag agc atg acc ctg aac ctggcc gac cca gtg ggg agc ctg aag 561 Lys Gln Ser Met Thr Leu Asn Leu AlaAsp Pro Val Gly Ser Leu Lys 35 40 45 50 ccg cac ctc cgc gcc aag aac tcggac ctc ctc acc tcg ccc gac gtg 609 Pro His Leu Arg Ala Lys Asn Ser AspLeu Leu Thr Ser Pro Asp Val 55 60 65 ggg ctg ctc aag ctg gcg tcg ccc gagctg gag cgc ctg ata atc cag 657 Gly Leu Leu Lys Leu Ala Ser Pro Glu LeuGlu Arg Leu Ile Ile Gln 70 75 80 tcc agc aac ggg cac atc acc acc acg ccgacc ccc acc cag ttc ctg 705 Ser Ser Asn Gly His Ile Thr Thr Thr Pro ThrPro Thr Gln Phe Leu 85 90 95 tgc ccc aag aac gtg aca gat gag cag gag gggttc gcc gag ggc ttc 753 Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly PheAla Glu Gly Phe 100 105 110 gtg cgc gcc ctg gcc gaa ctg cac agc cag aacacg ctg ccc agc gtc 801 Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn ThrLeu Pro Ser Val 115 120 125 130 acg tcg gcg gcg cag ccg gtc aac ggg gcaggc atg gtg gct ccc gcg 849 Thr Ser Ala Ala Gln Pro Val Asn Gly Ala GlyMet Val Ala Pro Ala 135 140 145 gta gcc tcg gtg gca ggg ggc agc ggc agcggc ggc ttc agc gcc agc 897 Val Ala Ser Val Ala Gly Gly Ser Gly Ser GlyGly Phe Ser Ala Ser 150 155 160 ctg cac agc gag ccg ccg gtc tac gca aacctc agc aac ttc aac cca 945 Leu His Ser Glu Pro Pro Val Tyr Ala Asn LeuSer Asn Phe Asn Pro 165 170 175 ggc gcg ctg agc agc ggc ggc ggg gcg ccctcc tac ggc gcg gcc ggc 993 Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro SerTyr Gly Ala Ala Gly 180 185 190 ctg gcc ttt ccc gcg caa ccc cag cag cagcag cag ccg ccg cac cac 1041 Leu Ala Phe Pro Ala Gln Pro Gln Gln Gln GlnGln Pro Pro His His 195 200 205 210 ctg ccc cag cag atg ccc gtg cag cacccg cgg ctg cag gcc ctg aag 1089 Leu Pro Gln Gln Met Pro Val Gln His ProArg Leu Gln Ala Leu Lys 215 220 225 gag gag cct cag aca gtg ccc gag atgccc ggc gag aca ccg ccc ctg 1137 Glu Glu Pro Gln Thr Val Pro Glu Met ProGly Glu Thr Pro Pro Leu 230 235 240 tcc ccc atc gac atg gag tcc cag gagcgg atc aag gcg gag agg aag 1185 Ser Pro Ile Asp Met Glu Ser Gln Glu ArgIle Lys Ala Glu Arg Lys 245 250 255 cgc atg agg aac cgc atc gct gcc tccaag tgc cga aaa agg aag ctg 1233 Arg Met Arg Asn Arg Ile Ala Ala Ser LysCys Arg Lys Arg Lys Leu 260 265 270 gag aga atc gcc cgg ctg gag gaa aaagtg aaa acc ttg aaa gct cag 1281 Glu Arg Ile Ala Arg Leu Glu Glu Lys ValLys Thr Leu Lys Ala Gln 275 280 285 290 aac tcg gag ctg gcg tcc acg gccaac atg ctc agg gaa cag gtg gca 1329 Asn Ser Glu Leu Ala Ser Thr Ala AsnMet Leu Arg Glu Gln Val Ala 295 300 305 cag ctt aaa cag aaa gtc atg aaccac gtt aac agt ggg tgc caa ctc 1377 Gln Leu Lys Gln Lys Val Met Asn HisVal Asn Ser Gly Cys Gln Leu 310 315 320 atg cta acg cag cag ttg caa acattt tgaagagaga ccgtcggggg 1424 Met Leu Thr Gln Gln Leu Gln Thr Phe 325330 ctgaggggca acgaagaaaa aaaataacac agagagacag acttgagaac ttgacaagtt1484 gcgacggaga gaaaaaagaa gtgtccgaga actaaagcca agggtatcca agttggactg1544 ggttcggtct gacggcgccc ccagtgtgca cgagtgggaa ggacctggtc gcgccctccc1604 ttggcgtgga gccagggagc ggccgcctgc gggctgcccc gctttgcgga cgggctgtcc1664 ccgcgcgaac ggaacgttgg actttcgtta acattgacca agaactgcat ggacctaaca1724 ttcgatctca ttcagtatta aagggggcag ggggaggggg ttacaaactg caatagagac1784 tgtagattgc ttctgtagta ctccttaaga acacaaagcg gggggagggt tggggagggg1844 cggcaggagg gaggtttgtg agagcgaggc tgagcctaca gatgaactct ttctggcctg1904 ctttcgttaa ctgtgtatgt acatatatat attttttaat ttgattaaag ctgattactg1964 tcaataaaca gcttcatgcc tttgtaagtt atttcttgtt tgtttgtttg ggatcctgcc2024 cagtgttgtt tgtaaataag agatttggag cactctgagt ttaccatttg taataaagta2084 tataattttt tt 2096 10 331 PRT Homo sapiens 10 Met Thr Ala Lys MetGlu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala 1 5 10 15 Ser Phe Leu ProSer Glu Ser Gly Pro Tyr Gly Tyr Ser Asn Pro Lys 20 25 30 Ile Leu Lys GlnSer Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser 35 40 45 Leu Lys Pro HisLeu Arg Ala Lys Asn Ser Asp Leu Leu Thr Ser Pro 50 55 60 Asp Val Gly LeuLeu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile 65 70 75 80 Ile Gln SerSer Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln 85 90 95 Phe Leu CysPro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu 100 105 110 Gly PheVal Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro 115 120 125 SerVal Thr Ser Ala Ala Gln Pro Val Asn Gly Ala Gly Met Val Ala 130 135 140Pro Ala Val Ala Ser Val Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser 145 150155 160 Ala Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe165 170 175 Asn Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr GlyAla 180 185 190 Ala Gly Leu Ala Phe Pro Ala Gln Pro Gln Gln Gln Gln GlnPro Pro 195 200 205 His His Leu Pro Gln Gln Met Pro Val Gln His Pro ArgLeu Gln Ala 210 215 220 Leu Lys Glu Glu Pro Gln Thr Val Pro Glu Met ProGly Glu Thr Pro 225 230 235 240 Pro Leu Ser Pro Ile Asp Met Glu Ser GlnGlu Arg Ile Lys Ala Glu 245 250 255 Arg Lys Arg Met Arg Asn Arg Ile AlaAla Ser Lys Cys Arg Lys Arg 260 265 270 Lys Leu Glu Arg Ile Ala Arg LeuGlu Glu Lys Val Lys Thr Leu Lys 275 280 285 Ala Gln Asn Ser Glu Leu AlaSer Thr Ala Asn Met Leu Arg Glu Gln 290 295 300 Val Ala Gln Leu Lys GlnLys Val Met Asn His Val Asn Ser Gly Cys 305 310 315 320 Gln Leu Met LeuThr Gln Gln Leu Gln Thr Phe 325 330 11 1418 DNA Homo sapiens CDS(19)..(1170) 11 cattaattgc ttgccatc atg agc aga agc aag cgt gac aac aatttt tat 51 Met Ser Arg Ser Lys Arg Asp Asn Asn Phe Tyr 1 5 10 agt gtagag att gga gat tct aca ttc aca gtc ctg aaa cga tat cag 99 Ser Val GluIle Gly Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln 15 20 25 aat tta aaacct ata ggc tca gga gct caa gga ata gta tgc gca gct 147 Asn Leu Lys ProIle Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala 30 35 40 tat gat gcc attctt gaa aga aat gtt gca atc aag aag cta agc cga 195 Tyr Asp Ala Ile LeuGlu Arg Asn Val Ala Ile Lys Lys Leu Ser Arg 45 50 55 cca ttt cag aat cagact cat gcc aag cgg gcc tac aga gag cta gtt 243 Pro Phe Gln Asn Gln ThrHis Ala Lys Arg Ala Tyr Arg Glu Leu Val 60 65 70 75 ctt atg aaa tgt gttaat cac aaa aat ata att ggc ctt ttg aat gtt 291 Leu Met Lys Cys Val AsnHis Lys Asn Ile Ile Gly Leu Leu Asn Val 80 85 90 ttc aca cca cag aaa tcccta gaa gaa ttt caa gat gtt tac ata gtc 339 Phe Thr Pro Gln Lys Ser LeuGlu Glu Phe Gln Asp Val Tyr Ile Val 95 100 105 atg gag ctc atg gat gcaaat ctt tgc caa gtg att cag atg gag cta 387 Met Glu Leu Met Asp Ala AsnLeu Cys Gln Val Ile Gln Met Glu Leu 110 115 120 gat cat gaa aga atg tcctac ctt ctc tat cag atg ctg tgt gga atc 435 Asp His Glu Arg Met Ser TyrLeu Leu Tyr Gln Met Leu Cys Gly Ile 125 130 135 aag cac ctt cat tct gctgga att att cat cgg gac tta aag ccc agt 483 Lys His Leu His Ser Ala GlyIle Ile His Arg Asp Leu Lys Pro Ser 140 145 150 155 aat ata gta gta aaatct gat tgc act ttg aag att ctt gac ttc ggt 531 Asn Ile Val Val Lys SerAsp Cys Thr Leu Lys Ile Leu Asp Phe Gly 160 165 170 ctg gcc agg act gcagga acg agt ttt atg atg acg cct tat gta gtg 579 Leu Ala Arg Thr Ala GlyThr Ser Phe Met Met Thr Pro Tyr Val Val 175 180 185 act cgc tac tac agagca ccc gag gtc atc ctt ggc atg ggc tac aag 627 Thr Arg Tyr Tyr Arg AlaPro Glu Val Ile Leu Gly Met Gly Tyr Lys 190 195 200 gaa aac gtg gat ttatgg tct gtg ggg tgc att atg gga gaa atg gtt 675 Glu Asn Val Asp Leu TrpSer Val Gly Cys Ile Met Gly Glu Met Val 205 210 215 tgc cac aaa atc ctcttt cca gga agg gac tat att gat cag tgg aat 723 Cys His Lys Ile Leu PhePro Gly Arg Asp Tyr Ile Asp Gln Trp Asn 220 225 230 235 aaa gtt att gaacag ctt gga aca cca tgt cct gaa ttc atg aag aaa 771 Lys Val Ile Glu GlnLeu Gly Thr Pro Cys Pro Glu Phe Met Lys Lys 240 245 250 ctg caa cca acagta agg act tac gtt gaa aac aga cct aaa tat gct 819 Leu Gln Pro Thr ValArg Thr Tyr Val Glu Asn Arg Pro Lys Tyr Ala 255 260 265 gga tat agc tttgag aaa ctc ttc cct gat gtc ctt ttc cca gct gac 867 Gly Tyr Ser Phe GluLys Leu Phe Pro Asp Val Leu Phe Pro Ala Asp 270 275 280 tca gaa cac aacaaa ctt aaa gcc agt cag gca agg gat ttg tta tcc 915 Ser Glu His Asn LysLeu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser 285 290 295 aaa atg ctg gtaata gat gca tct aaa agg atc tct gta gat gaa gct 963 Lys Met Leu Val IleAsp Ala Ser Lys Arg Ile Ser Val Asp Glu Ala 300 305 310 315 ctc caa cacccg tac atc aat gtc tgg tat gat cct tct gaa gca gaa 1011 Leu Gln His ProTyr Ile Asn Val Trp Tyr Asp Pro Ser Glu Ala Glu 320 325 330 gct cca ccacca aag atc cct gac aag cag tta gat gaa agg gaa cac 1059 Ala Pro Pro ProLys Ile Pro Asp Lys Gln Leu Asp Glu Arg Glu His 335 340 345 aca ata gaagag tgg aaa gaa ttg ata tat aag gaa gtt atg gac ttg 1107 Thr Ile Glu GluTrp Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Leu 350 355 360 gag gag agaacc aag aat gga gtt ata cgg ggg cag ccc tct cct tta 1155 Glu Glu Arg ThrLys Asn Gly Val Ile Arg Gly Gln Pro Ser Pro Leu 365 370 375 gca cag gtgcag cag tgatcaatgg ctctcagcat ccatcatcat cgtcgtctgt 1210 Ala Gln Val GlnGln 380 caatgatgtg tcttcaatgt caacagatcc gactttggcc tctgatacagacagcagtct 1270 agaagcagca gctgggcctc tgggctgctg tagatgacta cttgggccatcggggggtgg 1330 gagggatggg gagtcggtta gtcattgata gaactacttt gaaaacaattcagtggtctt 1390 atttttgggt gatttttcaa aaaatgta 1418 12 384 PRT Homosapiens 12 Met Ser Arg Ser Lys Arg Asp Asn Asn Phe Tyr Ser Val Glu IleGly 1 5 10 15 Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Asn Leu LysPro Ile 20 25 30 Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Tyr Asp AlaIle Leu 35 40 45 Glu Arg Asn Val Ala Ile Lys Lys Leu Ser Arg Pro Phe GlnAsn Gln 50 55 60 Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Met LysCys Val 65 70 75 80 Asn His Lys Asn Ile Ile Gly Leu Leu Asn Val Phe ThrPro Gln Lys 85 90 95 Ser Leu Glu Glu Phe Gln Asp Val Tyr Ile Val Met GluLeu Met Asp 100 105 110 Ala Asn Leu Cys Gln Val Ile Gln Met Glu Leu AspHis Glu Arg Met 115 120 125 Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly IleLys His Leu His Ser 130 135 140 Ala Gly Ile Ile His Arg Asp Leu Lys ProSer Asn Ile Val Val Lys 145 150 155 160 Ser Asp Cys Thr Leu Lys Ile LeuAsp Phe Gly Leu Ala Arg Thr Ala 165 170 175 Gly Thr Ser Phe Met Met ThrPro Tyr Val Val Thr Arg Tyr Tyr Arg 180 185 190 Ala Pro Glu Val Ile LeuGly Met Gly Tyr Lys Glu Asn Val Asp Leu 195 200 205 Trp Ser Val Gly CysIle Met Gly Glu Met Val Cys His Lys Ile Leu 210 215 220 Phe Pro Gly ArgAsp Tyr Ile Asp Gln Trp Asn Lys Val Ile Glu Gln 225 230 235 240 Leu GlyThr Pro Cys Pro Glu Phe Met Lys Lys Leu Gln Pro Thr Val 245 250 255 ArgThr Tyr Val Glu Asn Arg Pro Lys Tyr Ala Gly Tyr Ser Phe Glu 260 265 270Lys Leu Phe Pro Asp Val Leu Phe Pro Ala Asp Ser Glu His Asn Lys 275 280285 Leu Lys Ala Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu Val Ile 290295 300 Asp Ala Ser Lys Arg Ile Ser Val Asp Glu Ala Leu Gln His Pro Tyr305 310 315 320 Ile Asn Val Trp Tyr Asp Pro Ser Glu Ala Glu Ala Pro ProPro Lys 325 330 335 Ile Pro Asp Lys Gln Leu Asp Glu Arg Glu His Thr IleGlu Glu Trp 340 345 350 Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Leu GluGlu Arg Thr Lys 355 360 365 Asn Gly Val Ile Arg Gly Gln Pro Ser Pro LeuAla Gln Val Gln Gln 370 375 380 13 17 DNA Artificial sequence PCR primer13 caymgngayn tnaarcc 17 14 22 DNA Artificial sequence PCR primer 14gagagcccat nswccadatr tc 22 15 8 PRT Artificial Sequence Epitope tag 15Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 16 4 PRT Artificial sequenceConsensus sequence motif 16 Xaa Ser Pro Xaa 1 17 1780 DNA Homo sapiensCDS (59)..(1330) 17 gggcgggcga gggatctgaa acttgcccac ccttcgggatattgcaggac gctgcatc 58 atg agc gac agt aaa tgt gac agt cag ttt tat agtgtg caa gtg gca 106 Met Ser Asp Ser Lys Cys Asp Ser Gln Phe Tyr Ser ValGln Val Ala 1 5 10 15 gac tca acc ttc act gtc cta aaa cgt tac cag cagctg aaa cca att 154 Asp Ser Thr Phe Thr Val Leu Lys Arg Tyr Gln Gln LeuLys Pro Ile 20 25 30 ggc tct ggg gcc caa ggg att gtt tgt gct gca ttt gataca gtt ctt 202 Gly Ser Gly Ala Gln Gly Ile Val Cys Ala Ala Phe Asp ThrVal Leu 35 40 45 ggg ata agt gtt gca gtc aag aaa cta agc cgt cct ttt cagaac caa 250 Gly Ile Ser Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln AsnGln 50 55 60 act cat gca aag aga gct tat cgt gaa ctt gtc ctc tta aaa tgtgtc 298 Thr His Ala Lys Arg Ala Tyr Arg Glu Leu Val Leu Leu Lys Cys Val65 70 75 80 aat cat aaa aat ata att agt ttg tta aat gtg ttt aca cca caaaaa 346 Asn His Lys Asn Ile Ile Ser Leu Leu Asn Val Phe Thr Pro Gln Lys85 90 95 act cta gaa gaa ttt caa gat gtg tat ttg gtt atg gaa tta atg gat394 Thr Leu Glu Glu Phe Gln Asp Val Tyr Leu Val Met Glu Leu Met Asp 100105 110 gct aac tta tgt cag gtt att cac atg gag ctg gat cat gaa aga atg442 Ala Asn Leu Cys Gln Val Ile His Met Glu Leu Asp His Glu Arg Met 115120 125 tcc tac ctt ctt tac cag atg ctt tgt ggt att aaa cat ctg cat tca490 Ser Tyr Leu Leu Tyr Gln Met Leu Cys Gly Ile Lys His Leu His Ser 130135 140 gct ggt ata att cat aga gat ttg aag cct agc aac att gtt gtg aaa538 Ala Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Ile Val Val Lys 145150 155 160 tca gac tgc acc ctg aag atc ctt gac ttt ggc ctg gcc cgg acagcg 586 Ser Asp Cys Thr Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala165 170 175 tgc act aac ttc atg atg acc cct tac gtg gtg aca cgg tac taccgg 634 Cys Thr Asn Phe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg180 185 190 gcg ccc gaa gtc atc ctg ggt atg ggc tac aaa gag aac gtt gatatc 682 Ala Pro Glu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile195 200 205 tgg tca gtg ggt tgc atc atg gga gag ctg gtg aaa ggt tgt gtgata 730 Trp Ser Val Gly Cys Ile Met Gly Glu Leu Val Lys Gly Cys Val Ile210 215 220 ttc caa ggc act gac cat att gat cag tgg aat aaa gtt att gagcag 778 Phe Gln Gly Thr Asp His Ile Asp Gln Trp Asn Lys Val Ile Glu Gln225 230 235 240 ctg gga aca cca tca gca gag ttc atg aag aaa ctt cag ccaact gtg 826 Leu Gly Thr Pro Ser Ala Glu Phe Met Lys Lys Leu Gln Pro ThrVal 245 250 255 agg aat tat gtc gaa aac aga cca aag tat cct gga atc aaattt gaa 874 Arg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Pro Gly Ile Lys PheGlu 260 265 270 gaa ctc ttt cca gat tgg ata ttc cca tca gaa tct gag cgagac aaa 922 Glu Leu Phe Pro Asp Trp Ile Phe Pro Ser Glu Ser Glu Arg AspLys 275 280 285 ata aaa aca agt caa gcc aga gat ctg tta tca aaa atg ttagtg att 970 Ile Lys Thr Ser Gln Ala Arg Asp Leu Leu Ser Lys Met Leu ValIle 290 295 300 gat cct gac aag cgg atc tct gta gac gaa gct ctg cgt caccca tac 1018 Asp Pro Asp Lys Arg Ile Ser Val Asp Glu Ala Leu Arg His ProTyr 305 310 315 320 atc act gtt tgg tat gac ccc gcc gaa gca gaa gcc ccacca cct caa 1066 Ile Thr Val Trp Tyr Asp Pro Ala Glu Ala Glu Ala Pro ProPro Gln 325 330 335 att tat gat gcc cag ttg gaa gaa aga gaa cat gca attgaa gaa tgg 1114 Ile Tyr Asp Ala Gln Leu Glu Glu Arg Glu His Ala Ile GluGlu Trp 340 345 350 aaa gag cta att tac aaa gaa gtc atg gat tgg gaa gaaaga agc aag 1162 Lys Glu Leu Ile Tyr Lys Glu Val Met Asp Trp Glu Glu ArgSer Lys 355 360 365 aat ggt gtt gta aaa gat cag cct tca gat gca gca gtaagt agc aac 1210 Asn Gly Val Val Lys Asp Gln Pro Ser Asp Ala Ala Val SerSer Asn 370 375 380 gcc act cct tct cag tct tca tcg atc aat gac att tcatcc atg tcc 1258 Ala Thr Pro Ser Gln Ser Ser Ser Ile Asn Asp Ile Ser SerMet Ser 385 390 395 400 act gag cag acg ctg gcc tca gac aca gac agc agtctt gat gcc tcg 1306 Thr Glu Gln Thr Leu Ala Ser Asp Thr Asp Ser Ser LeuAsp Ala Ser 405 410 415 acg gga ccc ctt gaa ggc tgt cga tgataggttagaaatagcaa acctgtcagc 1360 Thr Gly Pro Leu Glu Gly Cys Arg 420attgaaggaa ctctcacctc cgtgggcctg aaatgcttgg gagttgatgg aaccaaatag 1420aaaaactcca tgttctgcat gtaagaaaca caatgccttg ccctattcag acctgatagg 1480attgcctgct tagatgataa aatgaggcag aatatgtctg aagaaaaaaa ttgcaagcca 1540cacttctaga gattttgttc aagatcattt caggtgagca gttagagtag gtgaatttgt 1600ttcaaattgt actagtgaca gtttctcatc atctgtaact gttgagatgt atgtgcatgt 1660gaccacaaat gcttgcttgg acttgcccat ctagcacttt ggaaatcagt atttaaatgc 1720caaataatct tccaggtagt gctgcttctg aagttatctc ttaatcctct taagtaattt 178018 424 PRT Homo sapiens 18 Met Ser Asp Ser Lys Cys Asp Ser Gln Phe TyrSer Val Gln Val Ala 1 5 10 15 Asp Ser Thr Phe Thr Val Leu Lys Arg TyrGln Gln Leu Lys Pro Ile 20 25 30 Gly Ser Gly Ala Gln Gly Ile Val Cys AlaAla Phe Asp Thr Val Leu 35 40 45 Gly Ile Ser Val Ala Val Lys Lys Leu SerArg Pro Phe Gln Asn Gln 50 55 60 Thr His Ala Lys Arg Ala Tyr Arg Glu LeuVal Leu Leu Lys Cys Val 65 70 75 80 Asn His Lys Asn Ile Ile Ser Leu LeuAsn Val Phe Thr Pro Gln Lys 85 90 95 Thr Leu Glu Glu Phe Gln Asp Val TyrLeu Val Met Glu Leu Met Asp 100 105 110 Ala Asn Leu Cys Gln Val Ile HisMet Glu Leu Asp His Glu Arg Met 115 120 125 Ser Tyr Leu Leu Tyr Gln MetLeu Cys Gly Ile Lys His Leu His Ser 130 135 140 Ala Gly Ile Ile His ArgAsp Leu Lys Pro Ser Asn Ile Val Val Lys 145 150 155 160 Ser Asp Cys ThrLeu Lys Ile Leu Asp Phe Gly Leu Ala Arg Thr Ala 165 170 175 Cys Thr AsnPhe Met Met Thr Pro Tyr Val Val Thr Arg Tyr Tyr Arg 180 185 190 Ala ProGlu Val Ile Leu Gly Met Gly Tyr Lys Glu Asn Val Asp Ile 195 200 205 TrpSer Val Gly Cys Ile Met Gly Glu Leu Val Lys Gly Cys Val Ile 210 215 220Phe Gln Gly Thr Asp His Ile Asp Gln Trp Asn Lys Val Ile Glu Gln 225 230235 240 Leu Gly Thr Pro Ser Ala Glu Phe Met Lys Lys Leu Gln Pro Thr Val245 250 255 Arg Asn Tyr Val Glu Asn Arg Pro Lys Tyr Pro Gly Ile Lys PheGlu 260 265 270 Glu Leu Phe Pro Asp Trp Ile Phe Pro Ser Glu Ser Glu ArgAsp Lys 275 280 285 Ile Lys Thr Ser Gln Ala Arg Asp Leu Leu Ser Lys MetLeu Val Ile 290 295 300 Asp Pro Asp Lys Arg Ile Ser Val Asp Glu Ala LeuArg His Pro Tyr 305 310 315 320 Ile Thr Val Trp Tyr Asp Pro Ala Glu AlaGlu Ala Pro Pro Pro Gln 325 330 335 Ile Tyr Asp Ala Gln Leu Glu Glu ArgGlu His Ala Ile Glu Glu Trp 340 345 350 Lys Glu Leu Ile Tyr Lys Glu ValMet Asp Trp Glu Glu Arg Ser Lys 355 360 365 Asn Gly Val Val Lys Asp GlnPro Ser Asp Ala Ala Val Ser Ser Asn 370 375 380 Ala Thr Pro Ser Gln SerSer Ser Ile Asn Asp Ile Ser Ser Met Ser 385 390 395 400 Thr Glu Gln ThrLeu Ala Ser Asp Thr Asp Ser Ser Leu Asp Ala Ser 405 410 415 Thr Gly ProLeu Glu Gly Cys Arg 420 19 227 PRT Yeast HOG1 19 Met Thr Thr Asn Glu GluIle Arg Thr Gln Phe Gly Thr Val Glu Ile 1 5 10 15 Thr Asn Asn Asp AsnPro Val Met Phe Leu Ser Thr Thr Leu Thr Ser 20 25 30 Gln Pro Ile Met LysSer Thr Ala Val Leu Thr Lys Leu His Leu Arg 35 40 45 Glu Leu Cys Gln AspIle Ser Pro Leu Glu Ile Phe Thr Gln Gly Thr 50 55 60 Asp His Arg Leu LeuThr Arg Pro Glu Lys Gln Phe Val Gln Phe Ile 65 70 75 80 Arg Leu Tyr ValVal Leu Ile Asn Glu Asn Asp Cys Ile Gln Asp Pro 85 90 95 Gln Gly Ser IleMet Thr Trp Gln Lys Asp Val Glu Ile Ala Phe Ala 100 105 110 Ile Glu GlyPro Lys His Val His Phe Ser Ile Ile Thr Asp Leu Ser 115 120 125 Lys AspVal Ile Asn Thr Ile Cys Ser Glu Asn Thr Leu Lys Phe Thr 130 135 140 SerLeu His Arg Asp Pro Ile Pro Ser Glu Arg Lys Thr Val Glu Pro 145 150 155160 Asp Val Glu Phe Pro Lys Thr Ala Ala Asp Ala Ser Ala Pro Tyr His 165170 175 Thr Asp Pro Val Ala Ala Lys Phe Trp His Phe Asn Asp Ala Asp Leu180 185 190 Pro Val Asp Thr Arg Val Met Met Ser Ile Leu Phe His Lys IleGly 195 200 205 Gly Ser Asp Gly Gln Ile Asp Ile Ser Ala Thr Phe Asp AspGln Val 210 215 220 Ala Ala Ala 225 20 243 PRT Yeast MPK1 20 Met Ala AspLys Ile Glu Arg His Thr Phe Lys Val Phe Asn Gln Asp 1 5 10 15 Ser AspPhe Leu Ile Glu His Tyr Ser Arg Phe Ala Glu Ala Glu Asp 20 25 30 Thr ThrThr Asn Val Ser Lys Thr Leu Leu Cys Ser Leu Lys Leu Arg 35 40 45 His PheArg Gly Thr Cys Tyr Asp Met Asp Ile Val Phe Tyr Pro Asp 50 55 60 Gly SerIle Asn Gly Leu Leu Tyr Glu Glu Cys Asp Met His Ile Lys 65 70 75 80 SerGly Gln Pro Thr Asp Ala His Tyr Gln Ser Phe Thr Ile Leu Tyr 85 90 95 IleAsp Val Leu Gly Leu Leu Asn Ala Gln Cys Gly Tyr Ser Glu Asn 100 105 110Pro Val Glu Asn Ser Gln Phe Leu Glu Ala Trp Ile Met Ser Tyr Gln 115 120125 Thr Lys Ala Ile Val Ala Leu Ala Phe Leu Gly Gly Pro Ile Lys Lys 130135 140 Val Asn Leu Asn Gln Ile Leu Gln Val Asp Glu Thr Leu Arg Arg Ile145 150 155 160 Gly Ser Lys Asn Gln Asp Ile His Gln Leu Gly Phe Ile ProLys Val 165 170 175 Pro Val Asn Tyr Asn Ala Asn Leu Glu Gln Ala Phe ProGln Thr Glu 180 185 190 Leu Ser Ile His Ala Asp Pro Val Cys Ser Glu LysPhe Glu Phe Ser 195 200 205 Phe Glu Ser Val Asn Asp Met Asp Leu Gln MetVal Ile Gln Gln Phe 210 215 220 Arg Leu Phe Val Arg Gln Pro Leu Leu GluGlu Arg Gln Leu Gln Leu 225 230 235 240 Gln Gln Gln 21 230 PRT YeastFUS3 21 Met Ala Arg Thr Ile Asp Ile Pro Ser Gln Lys Leu Val Asp Leu Glu1 5 10 15 Tyr Thr Ser Ile His Lys Pro Ser Gly Ile Lys Ile Gln Ser LysLys 20 25 30 Leu Phe Val Thr Thr Ile Ile Lys Leu Arg Tyr Phe His Glu GluSer 35 40 45 Ile Asp Lys Val Arg Pro Val Ile Asp Lys Leu Asn Ala Leu GluGlu 50 55 60 Thr Asp Gln Lys Asn Asn Gln Asn Ser Gly Phe Ser Thr Ser AspAsp 65 70 75 80 His Val Gln Phe Thr Ile Arg Ala Leu Ser Ile Gln Val IleIle Leu 85 90 95 Leu Leu Asn Asn Asp Val Cys Cys Leu Ala Ser Ser Asp SerArg Glu 100 105 110 Thr Leu Val Gly Phe Glu Ala Trp Ile Met Thr Phe GlnGlu Thr Thr 115 120 125 Ala Met Ile Cys Leu Ala Ser Gly Pro His His LeuTrp Leu Ile Leu 130 135 140 Val Ser Phe Glu Asp Asn Gln Ile Lys Ser LysArg Ala Lys Glu Ile 145 150 155 160 Ala Leu Met Arg Pro Pro Leu Pro TrpThr Val Trp Ser Lys Thr Asp 165 170 175 Leu Asn Pro Asp Met Ile Asp GlnPhe Asn Pro Asp Ala Ala Arg Leu 180 185 190 Ala Met Tyr His Asp Pro TyrLeu Asn Leu Asp Glu Phe Trp Lys Leu 195 200 205 Asp Asn Lys Ile Met ArgPro Glu Glu Glu Val Pro Met Leu Asp Met 210 215 220 Leu Asp Leu Lys ThrMet 225 230 22 221 PRT Yeast KSS1 22 Met Pro Lys Arg Ile Val Tyr Asn IleSer Ser Asp Phe Leu Lys Ser 1 5 10 15 Leu Leu Glu Tyr Val Ser Thr HisLys Pro Thr Gly Glu Ile Ile Glu 20 25 30 Asp Lys Pro Leu Phe Leu Thr LeuIle Lys Ile Leu His Phe Lys Glu 35 40 45 Thr Ile Phe Ile Gln Arg Pro AspPhe Asn Asn Glu Ile Gln Gln Thr 50 55 60 Asp His Arg Ser Thr Gln Met SerAsp Asp His Ile Gln Phe Ile Thr 65 70 75 80 Arg Ala Val Val Gly Ser AsnVal Ile Leu Leu Ile Asn Asn Asp Val 85 90 95 Cys Ile Ile Asp Glu Ala AlaAsp Asn Ser Glu Pro Thr Gly Gln Gln 100 105 110 Ser Gly Glu Ala Trp MetThr Ser Ala Lys Ser Arg Ala Met Val Cys 115 120 125 Leu Ala Leu Phe LeuArg Arg Pro Ile Arg His Leu Leu Leu Ile Phe 130 135 140 Gly Ile Ile HisSer Asp Asn Asp Leu Arg Cys Ile Glu Ser Arg Ala 145 150 155 160 Glu IleLys Ser Leu Met Pro Ala Ala Pro Leu Met Arg Val Asn Pro 165 170 175 LysGly Ile Gln Arg Phe Pro Ala Thr Ala Lys Glu Leu Gln Thr Tyr 180 185 190His Asn Asp Pro Gly Glu Pro Ser Phe Phe Glu Phe Asp His His Lys 195 200205 Glu Ala Leu Thr Lys Asp Leu Lys Trp Asn Ile Phe Ser 210 215 220 23242 PRT Yeast ERK1 23 Met Ala Ala Ala Ala Ala Gln Gly Gly Gly Gly GlyGlu Pro Arg Arg 1 5 10 15 Thr Glu Gly Val Gly Pro Gly Val Pro Gly GluVal Glu Met Val Lys 20 25 30 Gly Gln Pro Asp Gly Pro Thr Gln Gln Tyr GluTyr Met Ser Ser His 35 40 45 Val Arg Lys Thr Arg Ile Glu His Tyr Cys GlnThr Leu Ile Gln Ile 50 55 60 Leu Leu Arg Phe Arg Glu Val Ile Arg Asp IleLeu Arg Ala Ser Thr 65 70 75 80 Ala Met Arg Gln Asp Glu Thr Asp Tyr LysLeu Leu Lys Ser Gln Gln 85 90 95 Ser Asn Asp His Ile Cys Phe Ile Arg LeuTyr Ile Asn Val Leu Leu 100 105 110 Leu Ile Asn Thr Thr Asp Cys Ile AspPro Glu His Asp His Thr Gly 115 120 125 Phe Leu Glu Ala Trp Ile Met AsnSer Lys Thr Lys Ser Ile Ile Leu 130 135 140 Ala Leu Ser Asn Arg Pro IleLys His Leu Leu His Ile Leu Gly Ile 145 150 155 160 Ser Ser Gln Glu AspLeu Asn Cys Ile Ile Asn Met Lys Ala Asn Leu 165 170 175 Gln Ser Leu SerLys Thr Lys Val Ala Trp Ala Lys Ser Asp Lys Leu 180 185 190 Asp Arg ThrPhe Asn Pro Asn Thr Glu Ala Leu Glu Gln Tyr Thr Asp 195 200 205 Pro ValAla Glu Glu Pro Phe Thr Phe Ala Met Glu Leu Asp Asp Leu 210 215 220 ProLys Arg Leu Phe Gln Thr Ala Arg Phe Gln Pro Gly Val Leu Glu 225 230 235240 Ala Pro 24 220 PRT Yeast ERK2 24 Met Ala Ala Ala Ala Ala Ala Gly AlaGly Pro Glu Met Val Arg Gly 1 5 10 15 Gln Val Asp Gly Pro Thr Ser TyrGlu Tyr Met Ser Asn Val Asn Lys 20 25 30 Val Arg Ile Glu His Tyr Cys GlnThr Leu Ile Lys Ile Leu Leu Arg 35 40 45 Phe Arg Glu Ile Asn Asp Ile IleGln Ala Pro Thr Ile Gln Met Lys 50 55 60 Gln Asp Glu Thr Asp Tyr Lys LeuLeu Lys Thr Gln His Ser Asn Asp 65 70 75 80 His Ile Cys Phe Ile Arg LeuTyr Ile Asn Val Leu Leu Leu Leu Asn 85 90 95 Thr Thr Asp Cys Val Asp ProAsp His Asp His Thr Gly Phe Leu Glu 100 105 110 Ala Trp Ile Met Asn SerLys Thr Lys Ser Ile Ile Leu Ala Leu Ser 115 120 125 Asn Arg Pro Ile LysHis Leu Leu His Ile Leu Gly Ile Ser Ser Gln 130 135 140 Glu Asp Leu AsnCys Ile Ile Asn Leu Lys Ala Asn Leu Leu Ser Leu 145 150 155 160 His LysAsn Lys Val Pro Trp Asn Arg Asn Ala Asp Lys Leu Asp Thr 165 170 175 PheAsn Pro His Glu Glu Gln Ala Leu Glu Gln Tyr Asp Pro Ala Glu 180 185 190Ala Pro Phe Lys Phe Asp Met Glu Leu Asp Asp Leu Pro Lys Lys Leu 195 200205 Phe Glu Thr Ala Arg Phe Gln Pro Gly Tyr Arg Ser 210 215 220 25 81PRT Artificial sequence Consensus sequence 25 Gly Gly Ala Gly Val AlaVal Ala Ile Lys Lys Phe Arg Arg Glu His 1 5 10 15 Asn Tyr Leu Leu TyrGln Leu Lys His His Arg Asp Lys Pro Asn Cys 20 25 30 Leu Lys Asp Phe GlyLeu Ala Arg Thr Tyr Val Thr Arg Tyr Arg Ala 35 40 45 Pro Glu Leu Tyr AspTrp Ser Gly Cys Ile Glu Phe Gly Gln Gly Pro 50 55 60 Asp Leu Leu Met LeuLys Arg Ile Ala Leu His Pro Tyr Asp Pro Glu 65 70 75 80 Glu

What is claimed is:
 1. An isolated polynucleotide encoding a c-JunN-terminal kinase, wherein said polynucleotide hybridizes to SEQ ID No.13 and SEQ ID No. 14 under stringent conditions and wherein saidpolynucleotide encodes a polypeptide that phosphorylates the c-JunN-terminal activation domain.
 2. An isolated polynucleotide encoding ac-Jun N-terminal kinase, wherein said polynucleotide hybridizes to thecomplement of SEQ ID No. 11 under stringent conditions and wherein saidpolynucleotide encodes a polypeptide that phosphorylates the c-JunN-terminal activation domain.
 3. The polynucleotide of claim 1 or 2wherein the hybridization conditions comprise washing with 1×SSC, 0.05%SDS and 1 mM EDTA.
 4. The polynucleotide of claim 1 or 2 wherein thepolynucleotide is isolated from brain.
 5. The polynucleotide of claim 1or 2 wherein the polynucleotide is expressed in brain.
 6. An isolatednucleic acid encoding a c-Jun N-terminal kinase wherein said nucleicacid hybridizes to the complement of SEQ ID No. 11 under stringentconditions and wherein said nucleic acid encodes a polypeptide thatphosphorylates the c-Jun N-terminal activation domain.
 7. An isolatednucleic acid encoding a c-Jun N-terminal kinase wherein said nucleicacid hybridizes to the complement of SEQ ID No. 17 under stringentconditions and wherein said nucleic acid encodes a polypeptide thatphosphorylates the c-Jun N-terminal activation domain.
 8. The nucleicacid of claim 6 or 7 wherein the hybridization conditions comprisewashing 1×SSC, 0.05% SDS and 1 mM EDTA.
 9. The nucleic acid of claim 6or 7 wherein the nucleic acid is isolated from brain.
 10. The nucleicacid of claim 6 or 7 wherein the nucleic acid is expressed in brain. 11.A host cell containing the polynucleotide of claim 1 or
 2. 12. A hostcell containing the polynucleotide of claim
 4. 13. A host cellcontaining the polynucleotide of claim
 5. 14. A host cell containing thenucleic acid of claim 6 or
 7. 15. A host cell containing the nucleicacid of claim
 9. 16. A host cell containing the nucleic acid of claim10.
 17. An expression vector comprising a polynucleotide of claim 1 or2.
 18. An expression vector comprising the polynucleotide of claim 4.19. An expression vector comprising the polynucleotide of claim
 5. 20.An expression vector comprising the nucleic acid of claim 6 or
 7. 21. Anexpression vector comprising the nucleic acid of claim
 9. 22. Anexpression vector comprising the nucleic acid of claim
 10. 23. Anisolated polypeptide with serine threonine kinase activity that binds toresidues 33-79 of c-Jun (SEQ ID No. 1) and is capable of phosphorylatingthe c-Jun N-terminal activation domain.
 24. The polypeptide of claim 23wherein the polypeptide is expressed in brain.
 25. The polypeptide ofclaim 23 or 24 which is approximately 46 kD as determined by SDS-PAGE.26. The polypeptide of claim 23 or 24 which is approximately 55 kD asdetermined by SDS-PAGE.
 27. The polypeptide of claim 23 or 24 which isapproximately 46 kD or 55 kD as determined by SDS-PAGE.